Patent Publication Number: US-2021171959-A1

Title: Compositions and methods for use in controlling mosquito-borne viruses

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to U.S. provisional application No. 62/724,468, filed Aug. 29, 2018, the disclosure of which is incorporated herein by reference. 
    
    
     FIELD 
     The present disclosure relates generally to inhibiting transmission of mosquito-borne viruses, such as dengue (DENV), Zika (ZIKV) and Chikungunya (CHIKV) viruses, and more specifically to inhibition of expression of mosquito genes to achieve this inhibition. 
     BACKGROUND 
     The  Aedes aegypti  mosquito transmits a range of viruses between humans, including DENV, ZIKV and Chikungunya (CHIKV). These viruses cause significant morbidity and mortality globally, with dengue fever alone estimated to affect 96 million people each year 1 . Vaccines and anti-viral chemotherapies against these viruses are either currently unavailable or are limited in their efficacy 2,3 , while urbanisation, globalisation and the spread of insecticide resistance is making traditional methods of mosquito control increasingly difficult 3-6 . Consequently, novel strategies could play a major role in alleviating human populations from these pathogens 5 . One control strategy that may be used against these viruses is the use of a bacterium called  Wolbachia pipientis  that lives within insect cells.  Wolbachia  can ‘block’ the growth and transmission of viruses when introduced into the mosquito vector  Aedes aegypti.  Despite successful releases of  Wolbachia  into natural mosquito populations, it is unclear whether the blocking phenotype will remain stable over time. Thus, there is an ongoing need for improved compositions and methods for controlling these viruses that do not rely on use of  Wolbachia.  The present disclosure is pertinent to this need. 
     SUMMARY 
     The present disclosure provides compositions and methods for inhibiting transmission of viruses that use mosquitoes as vectors. In embodiments, the viruses are DENV, ZIKV, or CHIKV. In embodiments, the mosquitoes are  Aedes aegypti.  In embodiments, RNA interference (RNAi) is used to inhibit expression of one or more mosquito genes. In embodiments, the mosquito gene is alpha-mannosidase 2, or Cadherin87A, or expression of a combination of mosquito genes is inhibited. In embodiments, the mosquitoes may be infected by  Wolbachia  bacteria. In embodiments, the mosquitoes are not infected by  Wolbachia  bacteria. 
     RNAi inhibition can be achieved using a variety of RNAi agents and RNAi delivery techniques, such as by direct injection of an RNAi agent, administration of a recombinant vector encoding an RNAi agent, or by infecting the mosquitoes with bacteria that are modified to express the RNAi agent. Combinations of such approaches are included in the disclosure. 
     In a particular embodiment, the disclosure provides a method for reducing viral load in mosquitoes. The method comprises administering to the mosquitoes or mosquito larvae an RNAi agent that inhibits expression of mosquito alpha-mannosidase 2, or an RNAi agent that inhibits expression of mosquito Cadherin87A, or administering a combination of said RNAi agents, such that exposure of the mosquitoes to the virus results reduced viral load. The reduced viral load may be relative to any suitable control value, such as viral load in mosquitoes that are exposed to the virus but do not comprise the administered RNAi agent. 
     The disclosure also includes modified mosquitoes or mosquito larvae. The modified mosquitoes and/or the larvae comprise at least one administered or recombinantly expressed RNAi agent that inhibits expression of mosquito alpha-mannosidase 2, or mosquito Cadherin87A, or a combination of said RNAi agents. Such modified mosquitoes may be resistant to a viral infection and/or exhibit reduced capacity to transmit the viral infection to a mammalian host. 
     The disclosure also provides a method for inhibiting transmission of a virus between mammalian hosts. This approach comprises releasing the modified mosquitoes into a population of unmodified mosquitoes. 
     In another aspect, the disclosure provides an RNAi agent or an expression vector that encodes the RNAi agent, that can inhibit expression of  Aedes aegypti  alpha-mannosidase 2, or inhibit expression of  Aedes aegypti  Cadherin87A. Compositions comprising such RNAi agents are also included. 
    
    
     
       BRIEF DESCRIPTION OF FIGURES 
         FIG. 1 . Experimental design. Selection (High blocking and Low blocking) and control (Random blocking) treatments. Each treatment has 3 replicate lines and all lines are initiated from the same ancestral population of  Wolbachia -infected  Aedes aegypti  (wMel strain) mosquitoes derived from and repeatedly outcrossed with a population from Queensland, Australia. 
         FIG. 2 . The rapid evolution of  Wolbachia -mediated DENV blocking. a, Log 10  copies of DENV per mosquito in the High, Low and Random blocking treatments after 4 rounds of selection (G4). b, Log 10  copies of DENV per mosquito in the High, Low and Random blocking treatments every 2 generations of the selection experiment (G0, G2 and G4). Error bars: mean+−−1SE. c, Log 10  copies of DENV per mosquito in the evolved lines either treated with the antibiotic tetracycline to remove  Wolbachia  (Wolb−) or not (Wolb+). These mosquitoes were tested 4 generations after the selection experiment (G8). *P&lt;0.05, **P&lt;0.01, ***P&lt;0.001. 
         FIG. 3 .  Wolbachia  density correlates with strength of DENV blocking. The correlation between the log 10  copies of DENV per mosquito and the density of  Wolbachia  cells. Each point is an average for each line.  Wolbachia  density is the relative number of  Wolbachia  genome copies relative to mosquito genome copies (measured by amplification and quantification of the  Wolbachia  gene WD0513 and the  A. aegypti  gene RPS17 using qPCR.). 
         FIG. 4 . Manhattan plots showing significantly differentiated SNPs over the  A. aegypti  genome using the Cochran-Mantel-Haenszel (CMH) test. Pairwise comparison between: a, High and Low blocking populations; b, High and Random blocking populations; c, Low and Random populations; d, Ancestral and High populations; e, Ancestral and Low populations; f, Ancestral and Random populations. 
         FIG. 5 . Manhattan plots showing significantly differentiated SNPs over the  Wolbachia  genome using the Cochran-Mantel-Haenszel (CMH) test. Pairwise comparison between: a, High and Low blocking populations; b, High and Random blocking populations; c, Low and Random populations; d, Ancestral and High populations; e, Ancestral and Low populations; f, Ancestral and Random populations. 
         FIG. 6 . Correlation between the population growth rate (r) of  Wolbachia -infected mosquitoes and their ability to block DENV assuming low larval survival (43%). The population growth rate (r) is measured in the absence of DENV infection and thus is the intrinsic fitness of each mosquito population. It is calculated from an age-structured Leslie matrix model which combines different fitness measures. This output assumes low larval survival (43%), based upon unpublished data (Cator, L). The output under high larval survival (92%) is presented in  FIG. 10 . Hatch order was included as a random effect in the statistical analysis and so is represented here as separate lines of best fit. 
         FIG. 7 .  Wolbachia  removal with antibiotics.  Wolbachia  density in evolved lines that have been treated with the antibiotic tetracycline to cure them of  Wolbachia  (Wolb−) or not (Wolb+).  Wolbachia  density is measured by genome copies of  Wolbachia  (measured by amplification of the  Wolbachia  gene WD0513) relative to mosquito genome copies (measured by amplification of the  A. aegypti  gene RPS17). 
         FIG. 8 . There is no correlation between dengue virus load and  Wolbachia  density in mosquito ovaries.  Wolbachia  density is measured by genome copies of  Wolbachia  (measured by amplification of the  Wolbachia  gene WD0513) relative to mosquito genome copies. 
         FIG. 9 . Reference curve relating image detection of eggs (total particle area in Pixels 2 ) with a manual count of the eggs. Linear regression (Y=183.1547X-8596.8768). 
         FIG. 10 . Correlation between the population growth rate (r) of  Wolbachia -infected mosquitoes and their ability to block DENV assuming high larval survival (92%). The population growth rate (r) is measured in the absence of DENV infection and thus is the intrinsic fitness of each mosquito population. It is calculated from an age-structured Leslie matrix model which combines different fitness measures. This output assumes high (92%) larval survival, based upon unpublished data. Hatch order was included as a random effect in the statistical analysis and so is represented here as separate lines of best fit. 
         FIG. 11 . RNA silencing of Cadherin gene in mosquitoes. Significant effect on dengue virus load in the carcass of  Wolbachia  infected mosquitoes 14 days post feeding of virus via blood meal. Double stranded RNA designed to target GFP served as the negative control. W+/− indicates  Wolbachia  infection status. P value by Sidak&#39;s multiple comparisons test. 
         FIG. 12 . RNA silencing of alpha mannosidase gene in mosquitoes. Significant effect on dengue virus load in the midgut of  Wolbachia  free mosquitoes 10 days post feeding of virus via blood meal. Double stranded RNA designed to target GFP served as the negative control. W+/− indicates  Wolbachia  infection status. P value by Sidak&#39;s multiple comparisons test. 
     
    
    
     DETAILED DESCRIPTION OF DISCLOSURE 
     Unless defined otherwise herein, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. 
     Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein. 
     The disclosure includes the DNA equivalent of every RNA sequence described herein, and the RNA equivalent of every DNA sequence described herein. The disclosure includes all complementary sequences, as well as reverse complement sequences. 
     The disclosure includes all single-nucleotide polymorphisms (SNPs) described herein, and all manner of testing for such SNPs using any suitable sample obtained or derived from mosquitoes, as described further below. 
     The disclosure also includes sequences that have at from 80-99% identity with the sequences described herein, provided nucleotide or amino acid changes do not alter the function of the molecule in question so that it does not achieve its intended effect. Thus, polynucleotide sequences described herein may have nucleotide insertions, deletions, and mutations. The disclosure includes the proviso that any polynucleotide that occurs naturally in mosquitoes can be excluded from invention. Accordingly, the disclosure comprises in certain embodiments use of a segment of an artificially generated RNA that would target mosquito mRNA using one or more molecular-biology based approaches, including but not necessarily limited to by altering the copy number of the gene encoding the RNA, by using only a segment of the RNA, by overexpressing the RNA, or by expressing the RNA or a segment thereof as further described herein from an expression vector. Modified mosquitoes that express any polynucleotide, or exhibit increased amounts and/or have increased gene copy numbers, are included in the disclosure. 
     Non-limiting embodiments of the disclosure are illustrated using a dsRNA adapted from  Aedes aegypti  mRNA encoding cadherin, and separately an  Aedes aegypti  mRNA encoding alpha mannosidase. All of the description that relates to this example applies to each and every other RNA polynucleotide described herein, including but not necessarily limited to those sequences represented by the information presented in Table A, which provides genes, cDNA sequences, and other information regarding  Aedes aegypti.    
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE A 
               
               
                   
               
               
                   
                 No.of 
                   
                   
                 Min- 
                 Max- 
                   
               
               
                 Gene 
                 snps 
                 #nucs 
                 Snps per bp 
                 pvalue 
                 pvalue 
                 gene name 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 AAEL004389 
                 357 
                 7138 
                 19.99439776 
                 1.00E−100 
                 0.04994742 
                 Alpha 
               
               
                   
                   
                   
                   
                   
                   
                 mannosidase 
               
               
                 AAEL023845 
                 627 
                 9594 
                 15.30143541 
                 7.58E−97 
                 0.04941792 
                 cadherin 
               
               
                 AAEL018695 
                 267 
                 5470 
                 20.48689139 
                 8.16E−76 
                 0.04992507 
                   
               
               
                 AAEL014511 
                 210 
                 4375 
                 20.83333333 
                 7.34E−70 
                 0.04875134 
                 N/A 
               
               
                 AAEL006196 
                 35 
                 2836 
                 81.02857143 
                 1.07E−68 
                 0.04758416 
                 hemomucin 
               
               
                 AAEL022656 
                 65 
                 1139 
                 17.52307692 
                 7.98E−68 
                 0.04167159 
                 N/A 
               
               
                 AAEL023759 
                 32 
                 3832 
                 119.75 
                 1.23E−66 
                 0.04845109 
                 N/A 
               
               
                 AAEL008334 
                 217 
                 9624 
                 44.35023041 
                 2.97E−64 
                 0.04955478 
                 N/A 
               
               
                 AAEL002769 
                 327 
                 10154 
                 31.05198777 
                 1.72E−59 
                 0.04810721 
                   
               
               
                 AAEL014528 
                 36 
                 1968 
                 54.66666667 
                 2.31E−55 
                 0.048897 
                   
               
               
                 AAEL010881 
                 401 
                 1141 
                 2.845386534 
                 1.72E−54 
                 0.04979738 
                   
               
               
                 AAEL019422 
                 357 
                 5530 
                 15.49019608 
                 2.34E−53 
                 0.04965694 
                 N/A 
               
               
                 AAEL027527 
                 131 
                 5172 
                 39.48091603 
                 3.70E−53 
                 0.0492046 
                 N/A 
               
               
                 AAEL005813 
                 53 
                 4412 
                 83.24528302 
                 2.48E−52 
                 0.04113651 
                 NELF 
               
               
                 AAEL025611 
                 9 
                 4700 
                 522.2222222 
                 3.01E−52 
                 0.02686835 
                 N/A 
               
               
                 AAEL004233 
                 385 
                 3660 
                 9.506493506 
                 1.95E−51 
                 0.04952328 
                 N/A 
               
               
                 AAEL019927 
                 35 
                 2932 
                 83.77142857 
                 3.80E−51 
                 0.04424921 
                 N/A 
               
               
                 AAEL019447 
                 136 
                 4920 
                 36.17647059 
                 3.80E−51 
                 0.04673519 
                 N/A 
               
               
                 AAEL004396 
                 456 
                 3657 
                 8.019736842 
                 8.28E−50 
                 0.04974027 
                   
               
               
                 AAEL022231 
                 157 
                 714 
                 4.547770701 
                 8.28E−50 
                 0.04855324 
                 N/A 
               
               
                 AAEL002173 
                 29 
                 2483 
                 85.62068966 
                 5.67E−49 
                 0.04144512 
                 N/A 
               
               
                 AAEL002169 
                 39 
                 9999 
                 256.3846154 
                 2.93E−48 
                 0.049814 
                 N/A 
               
               
                 AAEL022176 
                 48 
                 2777 
                 57.85416667 
                 2.98E−48 
                 0.04664656 
                 N/A 
               
               
                 AAEL002876 
                 341 
                 8046 
                 23.59530792 
                 2.98E−48 
                 0.04946527 
                 N/A 
               
               
                 AAEL009466 
                 249 
                 8180 
                 32.85140562 
                 1.46E−47 
                 0.04844907 
                   
               
               
                 AAEL020340 
                 418 
                 5253 
                 12.56698565 
                 9.35E−47 
                 0.04911214 
                 N/A 
               
               
                 AAEL021067 
                 573 
                 4325 
                 7.547993019 
                 1.73E−46 
                 0.04969989 
                 N/A 
               
               
                 AAEL004700 
                 118 
                 2001 
                 16.95762712 
                 1.94E−46 
                 0.04773173 
                 cdkl1/4 
               
               
                 AAEL024283 
                 485 
                 6723 
                 13.86185567 
                 3.41E−45 
                 0.04962042 
                 N/A 
               
               
                 AAEL013026 
                 101 
                 2669 
                 26.42574257 
                 1.56E−44 
                 0.03990847 
                   
               
               
                 AAEL024147 
                 95 
                 3805 
                 40.05263158 
                 3.00E−44 
                 0.0391458 
                 N/A 
               
               
                 AAEL019752 
                 427 
                 8435 
                 19.75409836 
                 5.50E−44 
                 0.04994045 
                 N/A 
               
               
                 AAEL010513 
                 240 
                 4144 
                 17.26666667 
                 6.20E−44 
                 0.04989911 
                   
               
               
                 AAEL019639 
                 146 
                 6661 
                 45.62328767 
                 1.30E−43 
                 0.04783913 
                 N/A 
               
               
                 AAEL020614 
                 198 
                 5633 
                 28.44949495 
                 3.64E−43 
                 0.04969003 
                 N/A 
               
               
                 AAEL019720 
                 98 
                 9507 
                 97.01020408 
                 1.02E−42 
                 0.0457651 
                 N/A 
               
               
                 AAEL005802 
                 16 
                 3863 
                 241.4375 
                 2.67E−42 
                 0.04815738 
                   
               
               
                 AAEL014044 
                 29 
                 2815 
                 97.06896552 
                 3.16E−42 
                 0.04769956 
                 N/A 
               
               
                 AAEL019638 
                 281 
                 6518 
                 23.19572954 
                 6.63E−42 
                 0.04991624 
                 N/A 
               
               
                 AAEL018306 
                 225 
                 5292 
                 23.52 
                 1.07E−41 
                 0.04928079 
                 N/A 
               
               
                 AAEL024508 
                 15 
                 3343 
                 222.8666667 
                 1.83E−41 
                 0.000932615 
                 N/A 
               
               
                 AAEL008617 
                 67 
                 8005 
                 119.4776119 
                 1.29E−40 
                 0.04903814 
                 N/A 
               
               
                 AAEL020230 
                 277 
                 5162 
                 18.63537906 
                 5.29E−40 
                 0.04854234 
                 N/A 
               
               
                 AAEL025250 
                 569 
                 5397 
                 9.485061511 
                 1.21E−39 
                 0.04994368 
                 N/A 
               
               
                 AAEL007483 
                 85 
                 3889 
                 45.75294118 
                 1.22E−39 
                 0.04357599 
                 N/A 
               
               
                 AAEL010721 
                 43 
                 2580 
                 60 
                 3.86E−39 
                 0.04873577 
                   
               
               
                 AAEL002879 
                 99 
                 8839 
                 89.28282828 
                 4.88E−39 
                 0.04611968 
                   
               
               
                 AAEL023695 
                 99 
                 5972 
                 60.32323232 
                 6.68E−39 
                 0.04794791 
                 N/A 
               
               
                 AAEL013723 
                 1129 
                 10081 
                 8.929140833 
                 7.57E−39 
                 0.04993501 
                   
               
               
                 AAEL019426 
                 363 
                 5999 
                 16.5261708 
                 1.80E−38 
                 0.04970021 
                 N/A 
               
               
                 AAEL002571 
                 330 
                 6941 
                 21.03333333 
                 1.82E−38 
                 0.04968003 
                   
               
               
                 AAEL002859 
                 722 
                 10617 
                 14.70498615 
                 2.94E−38 
                 0.04980661 
                 N/A 
               
               
                 AAEL018215 
                 236 
                 7299 
                 30.9279661 
                 3.99E−38 
                 0.04928266 
                 N/A 
               
               
                 AAEL006208 
                 31 
                 3705 
                 119.516129 
                 4.80E−38 
                 0.02803775 
                 N/A 
               
               
                 AAEL006817 
                 20 
                 1406 
                 70.3 
                 7.44E−38 
                 0.005209389 
                 N/A 
               
               
                 AAEL019991 
                 18 
                 715 
                 39.72222222 
                 1.80E−37 
                 0.03445773 
                 N/A 
               
               
                 AAEL020617 
                 78 
                 10877 
                 139.4487179 
                 3.58E−37 
                 0.0486428 
                 N/A 
               
               
                 AAEL004572 
                 498 
                 7443 
                 14.94578313 
                 7.69E−37 
                 0.04988507 
                 N/A 
               
               
                 AAEL011522 
                 146 
                 2116 
                 14.49315068 
                 1.57E−36 
                 0.0476363 
                   
               
               
                 AAEL007205 
                 78 
                 4816 
                 61.74358974 
                 2.34E−36 
                 0.04866314 
               
               
                   
               
            
           
         
       
     
     The disclosure includes all polynucleotide and amino acid sequences described herein, including in the text, figures, tables, and any supplemental material that is part of this disclosure. Any reference to a database that includes an accession or gene or other number or alphanumeric indicator includes the sequences associated with the database entry as of the filing date of this application or patent. For example, in Table A, each alphanumeric designation under the Gene column provide is an alphanumeric identifier for a gene that is indexed in, for example, www.vectorbase.org. Querying this database for, for instance, AAEL004389, leads to a transcript table. In the transcript table, there are RefSeq links. Each of these links leads to a GenBank accession entry for an  Aedes aegypti  gene, such as alpha-mannosidase 2 mRNA sequence using the AAEL004389 designation, provided as a cDNA sequence. The same approach applies to the Cadherin-87A gene (AAEL023845). The database entry also provides the amino acid sequence encoded by the cDNA sequence. All of these cDNA sequences (and their corresponding mRNA sequences) for each gene in Table A, the amino acid sequences encoded by those genes, and any polynucleotide encoding the amino acid sequences, are incorporated herein by reference as they exist in the database on the filing date of this application or patent. Further, those skilled in the art will recognize alternative methods for accessing the sequences, and will recognize that all of these sequences are encompassed for use in any embodiment of this disclosure. 
     This disclosure also shows that a gene in the mosquito showing evolutionary change in association with both weakened and improved DENV blocking is the cadherin-87A gene (AAEL023845). Therefore any RNAi agent that could be designed to target the mRNA of this gene and affect its expression is encompassed by this disclosure for use in, for example, limiting viral replication. The same applies to the alpha-mannosidase gene. Accordingly, the present disclosure provides for inhibiting transmission and/or blocking replication or transmission of DENV, ZIKV, CHIKV, or any combination thereof, to mammalian hosts, and thereby includes inhibiting development of viral infections in the mammalian hosts, including but not necessarily limited to human hosts. 
     In particular embodiments, the disclosure relates to inhibiting expression of  Aedes aegypti  Cadherin87A, and/or  Aedes aegypti  alpha-mannosidase, or any gene or protein encoded by the gene described by way of Table A, using a polynucleotide targeted to a segment of such gene(s) and/or RNA(s) encoded by them. 
     In non-limiting embodiments, the disclosure provides compositions and methods that relate to use of engineered polynucleotides that can participate in RNAi-mediated inhibition, to inhibit translation of mRNA, and/or to degrade mRNA, that encodes  Aedes aegypti  protein(s). All mRNAs, including all splice variants, which encode an  Aedes aegypti  CAD or  Aedes aegypti  alpha-mannosidase, or any RNA or protein described in Table A, are included as targets of the RNAi agents of this disclosure. In non-limiting embodiments, an approach of this disclosure use of a segment of RNA or derivative thereof encoding the  Aedes aegypti  Cadherin87A (CAD) protein or the  Aedes aegypti  alpha-mannosidase protein for RNAi mediated gene expression inhibition. The disclosure includes targeting any RNA encoding the  Aedes aegypti  CAD protein, the sequence of which is as follows. 
                              Aedes aegypti  Cadherin 87A protein (CAD)           (SEQ ID NO: 1)           MIASTQKQQQRWTVLIPLLTIGFLIRTCHCNLPPIFTQDMNNLALP                   ETTPVGSVVYRLEGYDPEGGNVSFGLLGSDNFMVDPISGDVKVIKP                   LDREDQDTLSFSVTIKDRISTAGIDSENDNVVNVPITIIVLDENDN                   PPEFRNVPYETEVLEDAKPGTTVFSDILVTDRDTVGDNLIVNCIPQ                   PQNPDACEKFAIETLESGQDRLTASVVLKGRLDYNERMIYQILLEA                   TDGMFNATAGLEIHVKDVQNSAPVFQGSLAAVINEDSKIGTLVMMI                   HARDGDRGQPRKIVYELVTNPMDYFLLDRQTGELRTAKPLDKEALP                   DDTGLIILTVKARELIDGVPGNDNLTTATTQASITIRDVNDSPPMF                   NKKEYFVSLSENTAPGTPLPIEMSVHDPDVGENAVFSLRLNDVSEV                   FDVEPKLVTGSSQISIRVANGSLDYENPNQRKFIVLVIAEETQTNP                   KLSSTATLTVSITDSNDNRPIFEQDSYSTTVSETAHPGHLITTITA                   RDLDSGHFGDQGIRYSLSGTGAELFNVDPITGAITVADCPSVDNDN                   NKRRRRRRQIPSSDELTQDYPDMKRFNVSTDGRSGVLDRGVDYMAY                   KIYNSGESNEYRDVNVVAPPTVSSSWETSSLEESDSTPAIESEEYF                   TPSSTTTPIHSNEIQHRSDVGPGRAPCLDYENQSVYYLSYKATDDE                   GRGQTSVVSLRITLLDANDSPPVCESPLYRASVDEGATLFEPPLVI                   KARDPDVISEINYRIIGNEAITRHFEIDKRSGQLTISKSTALDVNH                   LKSENVFFAVEASDGLFTTLCNVNITIRDVNNHAPQFSREHYLASI                   EENFPIGTRVERLQAIDLDTGINAEIRYRIQQGSFDDFAIDNQTGV                   VTIARKLDYDRRNTYQMEIVAADLGTPSLSGTTTLTVSIINSNDKA                   PYFTPTTQRAEISEDAEVGTLVHTLVALDPDVASSEALDYAATEPI                   TAVDKDGKEVRDTEDFKDMFRIDRTGKVFVNRKLQRDDFAVIRITV                   LVTDTTAPSIQQGEGLLIITIIDVNEEPPLFVPPWTPADPRYRFQV                   LEEQPIGTILTTMQATDADSTVAEYRMTDNSHFEINNTTGLIRTKA                   RIDYEQTPTIQFNVTVVDTGIPQLTSTAEVTVDIINTNDNDPAFDE                   PEYEMSVVENAPTGTVVGIVSARDADSGPYGQITYSLVGDHSAASF                   AIDPDTGVITVRDGTTLDRERTlEIGLTAIATDRAPDGTSRSTTAP                   VTIKLLDENDNVPTFSQKIYHATVAENAALNPPAAILQVLATDPDE                   GAAGDVKYSIIGSDIENTFRLDANSGILYPYASLLGLDGNYRIQIE                   ARDGLGSGPHSDRAEIKIEIQSINQHRPIFIMPALSNATVEIPENL                   AMTDYLVMTVKANDSDEGTNGKVLYHLQVNNQNVQETDEFIINEMS                   GELRIRKPLNRKKQARFELILVARDQGTPAWFETLRFLTVLLVDVN                   ENHPEFPDASNPYRFFIAENSPRDIRIGKIQAYYDTPDPKIYYYMM                   LGNEDGAFYVDKTTGDIYTNKTLDREEADVYALYIKASKKQDLLIT                   ERDRMMMSTKKLERDSTVAKVVVITVLDVNDNPPVFKQDVYYAGVS                   SKAAINELVTIVNATDRDLGVNSTMELFISGSYLYKYGATKTTGSI                   VPSPFTISKDGRITTANYMAEYNQDRFILDIVAKEVESPERVATTK                   VYVVVIFNPEQLVRVILSRPPSEVHMERDEIISELSNATQKLIIVD                   EIRYHVDSLGRIRMDWCDMYFHAIDMSSQTIVSVEEILREIDAKYD                   FLQDYNAGFSIENVVPAYATNVQDEFDLALAAIIALLIVLFVGAVS                   FIVLCCCLKHWVITIPNETRRKDALIKKQIIEDLNTTENPLWIEQK                   LKLYEEQELTMQVFSEPELTQQQQQHHHQQQLNSSNNTSSSLASHQ                   NQHHHVMQQQEQALVLGLDRRDSYPELSQGGGDNTYATIQPRNYAS                   NLSSVLMGTSGIGGGGGGGSGNGAAPAGGLSGEMSDYATLRNSRAP                   SMYEFRGSTFQVQQLNGGPGGDQPDYVTELI            
The following is a representative cDNA encoding of the mRNA encoding the  Aedes aegypti  CAD protein.
 
     
       
         
           
               
               
            
               
                   
                   Aedes aegypti  cadherin-87A (LOC110674038), 
               
               
                   
                 transcript variant X4, mRNA (provided as 
               
               
                   
                 cDNA sequence): 
               
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
                 TAGCAAAACGTAGCTGCTCCGTTGGTTCAGACTACAGTTGACGTCG 
               
               
                   
               
               
                   
                 CGATTTCAACCCGATTGGATTGGCTTCCCTTCAATCCGGACAAAAA 
               
               
                   
               
               
                   
                 CTCGGAAGAAACGTAAACGCCGCTTTTCGAACAGAGCATCTTGGTT 
               
               
                   
               
               
                   
                 GCTTTTGGGGCCTCGTGAAGCTCGTGTCGCCGGATGAGAGGATTTG 
               
               
                   
               
               
                   
                 GAAATACAGCAACAATAGCAGGATAATCTCCATATCATTGGTTGAC 
               
               
                   
               
               
                   
                 TATGGTAGCTCGTCATCGCTGTCGCTGGCTCACTGGTGAGCAAGGG 
               
               
                   
               
               
                   
                 AGGAAGCGTGGTGGTGAATAATTCGATAGGTGCAATTTTCACGGTG 
               
               
                   
               
               
                   
                 ATTGCTCGAGTGGTCGATTGAGAAGGACTGGCTGGGAAAACCGGTT 
               
               
                   
               
               
                   
                 TTCCACCAATTCAGTGTCGATTGTCGAAAGAAACCGACAAACAGTA 
               
               
                   
               
               
                   
                 TCGTTGGGTTCGTTTTGTGTGGCGGAGTGGGTTGAGTGTCTGATTA 
               
               
                   
               
               
                   
                 GAAATAAAAGTGGAAGAATATCATCACTGGCAGTTATCTGTAACTG 
               
               
                   
               
               
                   
                 ATTCGTTGCAGGCGTCGGTACCGCACCTGGCTCCGCGAAGCAATAT 
               
               
                   
               
               
                   
                 CAGCTCCGCTGTTGATGAAAGTTTTGCTTTAAGTTCTTCAGCTCCA 
               
               
                   
               
               
                   
                 AGTTTCTGTTGTTGTTTGCCCGGTTGTTGTTGTCAGTGCTGGTGTT 
               
               
                   
               
               
                   
                 TCTCCCATCCCCGGAAACCGGTACCATTACATAAAGAGCAAAGTTC 
               
               
                   
               
               
                   
                 TTCGCCGTACACCCAAGGCTTGCAACCGCGAACACGATGCGATGCT 
               
               
                   
               
               
                   
                 AAATCCTAAGCCAGTCTTCGAGGCGTTCCACTAGGACATTTGTGCC 
               
               
                   
               
               
                   
                 TCCTTCGGGAAAGTGATCTGGCGTCGTCATGAATATTTTATACGCG 
               
               
                   
               
               
                   
                 CTACGACGAGTGTGCTTCGCTTTTGCGATTTCCTGTCAGTCTGTGC 
               
               
                   
               
               
                   
                 AAAAATAATATCCCACTCAATACAAGAGCAGAAGCAAAAAGCCCCA 
               
               
                   
               
               
                   
                 CAGTAAGAAAAATAGTAGCAAAGCAGCATATCATAATAGTCGTTAA 
               
               
                   
               
               
                   
                 GAATAAAGAAAATATAATTGAAACGTGTTTCCGAGCGAAAAGGGAA 
               
               
                   
               
               
                   
                 AAAGTGTTGCCTCGGCGAGAGTTGCACAAAAAGTGGAGGAAATTAA 
               
               
                   
               
               
                   
                 AAGAAGCTACTATTCTCGTAACGAAAAGCCAAGAAGCGTGGTTGGT 
               
               
                   
               
               
                   
                 TGTGCGAAGGAAAAAGTGAATGATTTATTCAGTGGATCGTCTCTCG 
               
               
                   
               
               
                   
                 GGTTCGTTGGAGGAAACGTGTAAGAGAAGAGCAGTCAGCAGCAAGG 
               
               
                   
               
               
                   
                 TGAAGATTGTGCGAAAACTGTAAATCAAGCGGAACGACGACGGCGA 
               
               
                   
               
               
                   
                 CGAATATGAATGCGAAAGTTGAAGTCGACGGCCAGGTCGTCTTCAT 
               
               
                   
               
               
                   
                 CAGCATCATCAGAGAAGTTGTGGGCTGTAGTGACGGGTGGTGTAAA 
               
               
                   
               
               
                   
                 GTGTAGGAGTCTGCTGGTAAAGCTGAGTTGTAGTGGTTTTGTTTTT 
               
               
                   
               
               
                   
                 ATCAAGAAAGGATTCCAAGAAAGAAGAAAAGAACATTTAAGGAGAG 
               
               
                   
               
               
                   
                 TAGTGTCTTTGGCGTTTGGAGCTTTGCCGGTGCGGAACCCAATTAG 
               
               
                   
               
               
                   
                 AGCAGCTAAAGAAAGATTCATCTTTCGTAATTCAATATCTCTAAAC 
               
               
                   
               
               
                   
                 TGAACGGAAGTGAACTAGAATTGTGTGTGTGTGGCAAGGACGACCA 
               
               
                   
               
               
                   
                 GGCGACGAAGCAGCCGCCATTCAGCAATGATAGCCTCCACCCAGAA 
               
               
                   
               
               
                   
                 GCAGCAACAGCGATGGACAGTTTTAATACCGCTCCTAACGATAGGG 
               
               
                   
               
               
                   
                 TTCCTGATTCGGACATGTCACTGCAACCTGCCGCCGATTTTCACGC 
               
               
                   
               
               
                   
                 AGGACATGAACAACTTGGCCCTGCCGGAGACAACTCCGGTGGGAAG 
               
               
                   
               
               
                   
                 CGTCGTTTACCGGCTGGAGGGTTACGATCCGGAGGGCGGTAACGTC 
               
               
                   
               
               
                   
                 TCGTTTGGGCTGCTCGGCTCGGACAACTTTATGGTGGACCCAATCA 
               
               
                   
               
               
                   
                 GTGGGGACGTCAAGGTGATAAAACCGCTGGACCGTGAGGACCAGGA 
               
               
                   
               
               
                   
                 CACCCTCTCCTTCTCGGTGACCATCAAGGATCGCATCAGCACCGCA 
               
               
                   
               
               
                   
                 GGAATCGATTCCGAGAACGACAACGTGGTCAACGTTCCCATCACGA 
               
               
                   
               
               
                   
                 TAATCGTCCTGGACGAAAACGACAACCCACCGGAATTTCGCAATGT 
               
               
                   
               
               
                   
                 TCCCTACGAAACAGAGGTCCTGGAGGACGCCAAGCCAGGCACCACC 
               
               
                   
               
               
                   
                 GTGTTCAGCGATATCCTGGTTACCGATCGGGACACCGTCGGAGATA 
               
               
                   
               
               
                   
                 ACCTGATCGTGAACTGTATTCCACAACCGCAGAACCCGGATGCTTG 
               
               
                   
               
               
                   
                 CGAAAAGTTCGCCATCGAAACCCTCGAAAGCGGTCAGGATCGACTA 
               
               
                   
               
               
                   
                 ACGGCTTCGGTGGTGCTGAAGGGTCGCCTAGACTACAACGAACGGA 
               
               
                   
               
               
                   
                 TGATCTACCAGATTCTGCTGGAGGCTACCGATGGGATGTTCAACGC 
               
               
                   
               
               
                   
                 CACGGCTGGACTGGAGATCCACGTGAAGGATGTTCAGAACAGTGCG 
               
               
                   
               
               
                   
                 CCGGTGTTCCAAGGATCGTTGGCGGCGGTAATCAACGAGGACAGCA 
               
               
                   
               
               
                   
                 AGATCGGGACGCTGGTGATGATGATCCACGCAAGGGATGGCGATCG 
               
               
                   
               
               
                   
                 GGGTCAACCGAGGAAGATTGTCTACGAATTAGTTACGAACCCAATG 
               
               
                   
               
               
                   
                 GATTACTTCTTGCTGGATCGTCAAACGGGTGAGCTACGCACGGCCA 
               
               
                   
               
               
                   
                 AACCACTCGACAAGGAAGCCCTTCCCGACGACACCGGGTTGATAAT 
               
               
                   
               
               
                   
                 CCTGACGGTTAAAGCTCGCGAGCTGATCGACGGAGTTCCCGGTAAT 
               
               
                   
               
               
                   
                 GACAATCTGACCACGGCAACAACACAAGCGTCGATCACGATTCGCG 
               
               
                   
               
               
                   
                 ATGTGAACGATTCTCCACCGATGTTCAACAAAAAGGAATACTTCGT 
               
               
                   
               
               
                   
                 ATCGCTGTCGGAGAATACGGCTCCGGGAACGCCACTTCCGATCGAA 
               
               
                   
               
               
                   
                 ATGAGCGTTCATGATCCGGATGTTGGAGAGAACGCTGTGTTTTCTC 
               
               
                   
               
               
                   
                 TACGCTTGAATGATGTTTCGGAAGTGTTCGATGTGGAGCCAAAATT 
               
               
                   
               
               
                   
                 GGTGACGGGATCGTCACAGATTAGTATTCGTGTAGCGAATGGTTCG 
               
               
                   
               
               
                   
                 CTGGATTACGAAAACCCTAACCAACGGAAGTTCATCGTATTGGTGA 
               
               
                   
               
               
                   
                 TCGCTGAAGAAACCCAGACGAACCCTAAGCTGTCATCGACAGCTAC 
               
               
                   
               
               
                   
                 TTTAACGGTGTCTATCACCGACTCGAATGACAACCGTCCGATCTTC 
               
               
                   
               
               
                   
                 GAGCAGGACTCGTACTCTACAACTGTATCGGAAACTGCTCATCCCG 
               
               
                   
               
               
                   
                 GTCATTTGATAACGACCATCACCGCCAGAGATCTCGACTCAGGTCA 
               
               
                   
               
               
                   
                 TTTCGGCGACCAAGGAATTCGGTATTCCTTGTCTGGAACGGGAGCC 
               
               
                   
               
               
                   
                 GAACTCTTCAACGTCGACCCGATAACCGGCGCTATAACGGTCGCTG 
               
               
                   
               
               
                   
                 ATTGCCCATCCGTAGACAACGACAACAACAAAAGACGTCGTCGGCG 
               
               
                   
               
               
                   
                 ACGTCAGATTCCTTCATCCGATGAGCTGACTCAAGACTACCCGGAT 
               
               
                   
               
               
                   
                 ATGAAACGTTTCAACGTGTCAACCGACGGACGTTCGGGCGTCCTAG 
               
               
                   
               
               
                   
                 ACCGTGGCGTAGACTATATGGCCTACAAGATCTACAACAGTGGCGA 
               
               
                   
               
               
                   
                 ATCGAACGAGTACCGAGACGTGAATGTCGTCGCACCTCCAACGGTT 
               
               
                   
               
               
                   
                 TCCAGCAGCTGGGAAACGTCCAGTTTGGAGGAAAGCGACTCCACCC 
               
               
                   
               
               
                   
                 CGGCCATCGAGTCGGAAGAATACTTCACGCCATCTAGCACCACCAC 
               
               
                   
               
               
                   
                 TCCCATCCACTCGAACGAAATCCAGCACCGTTCGGATGTGGGCCCA 
               
               
                   
               
               
                   
                 GGGCGAGCTCCTTGCTTGGACTACGAAAATCAATCGGTGTACTATC 
               
               
                   
               
               
                   
                 TGTCCTACAAGGCCACGGATGACGAGGGCCGGGGTCAAACGTCGGT 
               
               
                   
               
               
                   
                 AGTATCGCTCCGGATCACCCTTCTGGATGCGAACGATTCGCCGCCG 
               
               
                   
               
               
                   
                 GTGTGCGAGAGCCCTCTCTATAGGGCATCGGTCGACGAGGGAGCCA 
               
               
                   
               
               
                   
                 CCCTATTTGAGCCGCCGCTCGTCATCAAAGCCCGCGATCCGGACGT 
               
               
                   
               
               
                   
                 TATTTCGGAAATTAATTATCGCATAATTGGTAACGAAGCAATTACG 
               
               
                   
               
               
                   
                 CGCCATTTCGAAATCGACAAACGGTCCGGACAGTTGACCATCTCCA 
               
               
                   
               
               
                   
                 AGAGTACCGCCCTGGACGTGAACCATCTGAAGTCGGAAAACGTGTT 
               
               
                   
               
               
                   
                 CTTCGCCGTGGAGGCAAGCGATGGCCTCTTCACCACCCTGTGCAAC 
               
               
                   
               
               
                   
                 GTGAACATCACCATCCGGGACGTGAACAACCATGCACCGCAGTTCT 
               
               
                   
               
               
                   
                 CCCGGGAGCACTATCTTGCCTCGATCGAGGAGAACTTCCCGATTGG 
               
               
                   
               
               
                   
                 CACCCGAGTCGAACGTTTACAGGCAATCGATTTGGATACCGGCATC 
               
               
                   
               
               
                   
                 AACGCCGAGATCAGGTACCGCATCCAGCAGGGAAGCTTCGATGACT 
               
               
                   
               
               
                   
                 TTGCCATCGACAACCAAACCGGGGTGGTGACCATCGCCCGGAAGTT 
               
               
                   
               
               
                   
                 GGACTACGACCGGAGGAACACCTACCAGATGGAAATAGTGGCAGCG 
               
               
                   
               
               
                   
                 GATCTGGGCACCCCAAGTCTGTCGGGGACAACCACCCTGACGGTGA 
               
               
                   
               
               
                   
                 GCATCATCAATAGCAACGACAAAGCCCCGTACTTTACGCCGACTAC 
               
               
                   
               
               
                   
                 TCAGCGGGCGGAAATATCGGAGGATGCGGAAGTGGGAACGTTGGTC 
               
               
                   
               
               
                   
                 CACACGCTGGTGGCACTCGATCCGGATGTGGCGTCCAGCGAAGCGT 
               
               
                   
               
               
                   
                 TGGATTATGCGGCAACGGAACCCATCACGGCCGTTGACAAGGACGG 
               
               
                   
               
               
                   
                 AAAGGAGGTGCGGGACACGGAAGATTTCAAGGACATGTTCCGCATC 
               
               
                   
               
               
                   
                 GATCGGACCGGAAAGGTGTTCGTCAATCGGAAGCTGCAGCGGGATG 
               
               
                   
               
               
                   
                 ATTTTGCGGTGATCCGAATCACGGTTCTGGTAACGGACACAACCGC 
               
               
                   
               
               
                   
                 CCCATCGATTCAGCAGGGCGAAGGTCTCCTCATAATCACAATCATC 
               
               
                   
               
               
                   
                 GACGTAAATGAAGAGCCACCGCTGTTCGTGCCCCCGTGGACTCCGG 
               
               
                   
               
               
                   
                 CGGATCCCCGCTACCGGTTCCAGGTGCTGGAGGAACAACCGATCGG 
               
               
                   
               
               
                   
                 TACCATCCTGACGACGATGCAAGCAACAGATGCCGACTCGACCGTC 
               
               
                   
               
               
                   
                 GCCGAGTACCGGATGACAGATAACAGCCATTTCGAGATAAACAACA 
               
               
                   
               
               
                   
                 CAACAGGTCTGATCCGCACCAAAGCCCGTATCGATTACGAGCAAAC 
               
               
                   
               
               
                   
                 GCCAACGATCCAGTTCAACGTCACCGTGGTGGACACCGGAATCCCG 
               
               
                   
               
               
                   
                 CAGTTGACGTCCACCGCCGAAGTAACGGTCGACATCATCAACACCA 
               
               
                   
               
               
                   
                 ACGACAACGATCCGGCCTTCGACGAGCCTGAGTACGAAATGTCCGT 
               
               
                   
               
               
                   
                 CGTGGAAAACGCACCCACCGGAACGGTTGTGGGCATAGTTTCAGCG 
               
               
                   
               
               
                   
                 CGGGATGCCGACTCGGGACCGTATGGCCAAATCACCTACTCCCTGG 
               
               
                   
               
               
                   
                 TCGGTGACCACAGTGCTGCCAGCTTTGCCATCGATCCAGACACCGG 
               
               
                   
               
               
                   
                 AGTTATCACGGTGCGCGACGGCACAACCTTGGACCGTGAACGGACA 
               
               
                   
               
               
                   
                 ACGGAAATCGGCCTCACTGCCATTGCCACGGATCGGGCCCCGGATG 
               
               
                   
               
               
                   
                 GAACCAGCCGGTCGACCACCGCACCGGTTACCATCAAACTGCTGGA 
               
               
                   
               
               
                   
                 CGAGAACGACAATGTGCCGACCTTCTCGCAGAAGATTTATCACGCC 
               
               
                   
               
               
                   
                 ACGGTAGCGGAAAATGCGGCACTCAATCCACCGGCAGCAATCTTGC 
               
               
                   
               
               
                   
                 AGGTTTTGGCCACCGATCCGGACGAGGGCGCTGCTGGGGACGTGAA 
               
               
                   
               
               
                   
                 ATATAGCATCATCGGTAGCGATATTGAAAACACCTTCCGGCTGGAC 
               
               
                   
               
               
                   
                 GCAAACTCGGGCATCCTGTATCCGTACGCCAGTTTGCTGGGACTCG 
               
               
                   
               
               
                   
                 ACGGCAACTATCGCATCCAAATCGAGGCCCGCGATGGCCTAGGATC 
               
               
                   
               
               
                   
                 CGGACCTCACAGCGATCGGGCTGAAATTAAAATTGAAATACAAAGC 
               
               
                   
               
               
                   
                 ATCAACCAGCATCGTCCGATTTTCATCATGCCGGCACTGTCCAACG 
               
               
                   
               
               
                   
                 CAACGGTGGAAATCCCCGAGAATTTAGCGATGACGGATTATCTCGT 
               
               
                   
               
               
                   
                 GATGACGGTTAAAGCGAACGACAGCGACGAGGGAACGAACGGCAAA 
               
               
                   
               
               
                   
                 GTTTTGTACCATCTGCAGGTCAACAACCAGAACGTCCAGGAAACGG 
               
               
                   
               
               
                   
                 ACGAGTTCATCATCAACGAAATGTCCGGCGAACTGCGCATTCGCAA 
               
               
                   
               
               
                   
                 GCCCCTCAACCGCAAGAAGCAGGCCCGCTTCGAGTTGATCCTGGTG 
               
               
                   
               
               
                   
                 GCCCGGGACCAGGGTACCCCTGCGTGGTTCGAAACGCTCCGTTTCC 
               
               
                   
               
               
                   
                 TCACCGTACTGCTGGTCGACGTCAACGAAAACCACCCGGAGTTTCC 
               
               
                   
               
               
                   
                 GGACGCCTCAAACCCCTACAGGTTCTTCATCGCCGAGAACAGTCCT 
               
               
                   
               
               
                   
                 CGGGACATCCGCATCGGTAAAATCCAGGCCTATTACGACACACCCG 
               
               
                   
               
               
                   
                 ACCCGAAAATCTACTACTACATGATGCTCGGCAACGAGGATGGAGC 
               
               
                   
               
               
                   
                 GTTCTACGTGGACAAAACCACCGGCGATATCTACACCAACAAAACG 
               
               
                   
               
               
                   
                 CTGGACCGCGAGGAAGCGGATGTCTACGCTCTCTATATCAAAGCCA 
               
               
                   
               
               
                   
                 GCAAGAAACAAGACCTGCTGATCACTGAGCGCGATCGGATGATGAT 
               
               
                   
               
               
                   
                 GTCGACCAAAAAGCTGGAACGCGATAGCACGGTTGCGAAGGTCTGG 
               
               
                   
               
               
                   
                 ATCACAGTCCTCGATGTCAACGACAATCCCCCGGTCTTTAAACAGG 
               
               
                   
               
               
                   
                 ACGTTTACTACGCTGGCGTAAGCTCCAAGGCTGCCATCAACGAATT 
               
               
                   
               
               
                   
                 GGTGACAATTGTCAATGCGACCGATCGAGATCTGGGCGTGAACTCT 
               
               
                   
               
               
                   
                 ACCATGGAACTGTTCATCAGCGGGTCTTATCTTTACAAATACGGAG 
               
               
                   
               
               
                   
                 CTACGAAGACAACTGGTAGCATAGTTCCAAGTCCGTTCACTATTTC 
               
               
                   
               
               
                   
                 CAAGGACGGTCGTATAACTACCGCAAACTACATGGCCGAATATAAC 
               
               
                   
               
               
                   
                 CAGGACCGTTTCATTCTGGACATTGTAGCAAAAGAGGTGGAATCTC 
               
               
                   
               
               
                   
                 CTGAGCGAGTTGCCACCACCAAAGTCTACGTCTGGATCTTCAATCC 
               
               
                   
               
               
                   
                 AGAACAACTAGTGCGTGTGATCCTGTCGAGGCCACCCTCGGAAGTT 
               
               
                   
               
               
                   
                 CACATGGAGCGAGATGAGATCATATCCGAACTTTCGAATGCCACCC 
               
               
                   
               
               
                   
                 AGAAGCTGATTATTGTCGATGAGATTCGATACCACGTGGACAGCTT 
               
               
                   
               
               
                   
                 GGGTCGCATTCGGATGGATTGGTGCGACATGTACTTCCATGCGATC 
               
               
                   
               
               
                   
                 GATATGAGTTCGCAGACGATCGTGTCGGTAGAGGAGATTCTGCGGG 
               
               
                   
               
               
                   
                 AGATCGACGCCAAATATGATTTCCTACAGGATTACAATGCCGGCTT 
               
               
                   
               
               
                   
                 TTCGATCGAGAACGTAGTCCCGGCCTACGCAACCAACGTCCAGGAC 
               
               
                   
               
               
                   
                 GAGTTCGATTTGGCCCTGGCTGCGATAATCGCCCTGCTGATAGTGC 
               
               
                   
               
               
                   
                 TGTTTGTCGGTGCCGTAAGCTTCATCGTCCTGTGCTGCTGTCTCAA 
               
               
                   
               
               
                   
                 ACATTGGGTCATTACGATTCCGAACGAAACCAGAAGAAAGGACGCC 
               
               
                   
               
               
                   
                 TTGATCAAAAAGCAGATTATCGAAGATTTAAATACGACCGAGAATC 
               
               
                   
               
               
                   
                 CACTTTGGATCGAGCAAAAACTGAAGCTCTACGAAGAGCAGGAACT 
               
               
                   
               
               
                   
                 GACGATGCAAGTGTTTTCCGAGCCGGAACTGACGCAACAGCAGCAG 
               
               
                   
               
               
                   
                 CAGCACCACCACCAACAGCAGTTGAACAGCTCGAACAATACTTCGT 
               
               
                   
               
               
                   
                 CGTCGTTGGCCAGCCACCAGAACCAGCACCACCATGTGATGCAACA 
               
               
                   
               
               
                   
                 GCAGGAACAAGCGTTGGTCCTGGGGCTGGATCGGCGGGATTCGTAC 
               
               
                   
               
               
                   
                 CCGGAATTGTCCCAAGGGGGCGGCGATAACACGTACGCCACCATCC 
               
               
                   
               
               
                   
                 AGCCACGCAATTATGCGTCCAATCTGAGCTCGGTGCTGATGGGCAC 
               
               
                   
               
               
                   
                 TAGCGGGATTGGTGGCGGCGGCGGTGGCGGAAGCGGAAACGGTGCG 
               
               
                   
               
               
                   
                 GCCCCGGCAGGCGGACTGAGCGGAGAAATGTCGGATTATGCGACAC 
               
               
                   
               
               
                   
                 TGCGGAACAGCAGGGCACCCTCGATGTACGAGTTCCGAGGTTCAAC 
               
               
                   
               
               
                   
                 CTTCCAGGTACAGCAGCTAAACGGTGGACCCGGCGGTGACCAGCCA 
               
               
                   
               
               
                   
                 GACTACGTGACGGAACTGATTTAAGAGTAAACAACCTTCGAACAGC 
               
               
                   
               
               
                   
                 ATCGAACCGTTTTGACCCAACTCAGCCCCAAAAGTGCAACAGTGGA 
               
               
                   
               
               
                   
                 ACAAACCGTTTTACGCTCTCGAGATGGACAGAGAAAGAGAGAGCAA 
               
               
                   
               
               
                   
                 CATCACTTTTTGGGTTTTTAGCATAGGATATCATCAGGAGACTAGA 
               
               
                   
               
               
                   
                 AAGCGGTTTGGAATTTACAAACCAACCGGAATCGCCGGATTGCCAA 
               
               
                   
               
               
                   
                 TTTGGATTTGTAGAAAATGAATGCTCAATGTGTATGACACCCGAAT 
               
               
                   
               
               
                   
                 GAAATACTCAAGTGAAGGAAAAGTTCGGAAAGCGATTTTTTAAATT 
               
               
                   
               
               
                   
                 ACTGATGAGAGGCACAGATTACAAAACACTCTTTGATAGACAATAA 
               
               
                   
               
               
                   
                 ATAGGAGATATCTTAAAAGGATAGTATTTATGACGGAGGAAGCAAC 
               
               
                   
               
               
                   
                 ATTGAAGAGATAAACGCACCCGGAGAAAATTGAATCATTCCACACG 
               
               
                   
               
               
                   
                 CGTACTCATTCCGAGTTTAAGTTGTAATTAATTTAAGTTCACAAAA 
               
               
                   
               
               
                   
                 ATACATTAACAGATGACCACCAGAATCGAATTCGAGCTATCACGAC 
               
               
                   
               
               
                   
                 CCGACTCCCCCTTCATTTAAAGGTGCTCGATAGGCAGGGAGCGGAC 
               
               
                   
               
               
                   
                 GAGTGGCCATTTACTTCACTTGGATACCTCGGCGGTCTGGGGCCAG 
               
               
                   
               
               
                   
                 CGGCCATTTCGAGCTCATTATAATTTCTCCCATTTTCTGCCAATTA 
               
               
                   
               
               
                   
                 CCAACGAACGTTCGCTCCACCACACTCTCACACGTGGTCGGTACCG 
               
               
                   
               
               
                   
                 GCACGCGCTTGAGTTCAACAAATGAATGCAATTAAAAATTACGCAA 
               
               
                   
               
               
                   
                 AACGAGATTGGGGGAAAAATTCTGCGCACCAAAAGGCATCAATGTG 
               
               
                   
               
               
                   
                 CAATTTTTCGAGAGAGGCAGGAAGAATATACTGAAAGGGATAAGAG 
               
               
                   
               
               
                   
                 GTCGAATGTGTCGAAATAGTCGAAATCAAGCATTTTTCGAATGGGT 
               
               
                   
               
               
                   
                 TTCCCTACGAAAGGCGGAAATCACGAAAGGCTCAACTCGTAAAAGC 
               
               
                   
               
               
                   
                 TGAAAATATCAAAATACTGAATGGTATTCAAGTCTTCTCTAGTAAA 
               
               
                   
               
               
                   
                 TCTAGTTTCTAGATGTCATCTTGCAATTCAACTAGCCCGAATGACA 
               
               
                   
               
               
                   
                 CTAACTTGCAAGCATCTTATCCGAACTTTATGAACATTCAGCTTTT 
               
               
                   
               
               
                   
                 TTTGTTTCGGCCTTAAGTTGGAATCCTTCGGATGTTACTTTTCTAG 
               
               
                   
               
               
                   
                 GGTTGCACAGCATCCAGTGAACTGACAATGGCAGTTGAAGAGGCAC 
               
               
                   
               
               
                   
                 AATTTAATAATTTTGAAAATTACTTACAAATTCGTTGACATAAAAA 
               
               
                   
               
               
                   
                 AAACTCCGTGAGCTCACTAGAAAATTGTAAAAAATGTCGTTTTGAT 
               
               
                   
               
               
                   
                 ACAACCTGTATGAAAAATTTGTTTTAACTCAAAACTTATTACGAGT 
               
               
                   
               
               
                   
                 TTCAAAATCAATTCAGTTCAAATCAGCATCAAACGCCTCATTTCGT 
               
               
                   
               
               
                   
                 TCAATACTCTTTCGTTGAGAATATTTGTCTACCGTTCGCATTTGGA 
               
               
                   
               
               
                   
                 ATACAACTTTTATGTATTTCGACCTAAAGACCATTCGACCTATTAT 
               
               
                   
               
               
                   
                 CGCGATGCGAAACAACCCCCAACTGTGAAGTGAATATCAGCGCGTG 
               
               
                   
               
               
                   
                 TCGTTCGAAAAAAAAAGACTCAAAAATCCAACCGCCGACGCCAAAC 
               
               
                   
               
               
                   
                 CGTCGAGGTATCGAGAGTCGAACATTGTAAATAATTAAATCTAGCA 
               
               
                   
               
               
                   
                 AAATGGATGAGAATATTTAAATTATTAAAAATCAATTATGTTAACG 
               
               
                   
               
               
                   
                 AGTTTACAGAGACATGCGGGAGGGTGAGGGCTTCGAACAAGGGTCT 
               
               
                   
               
               
                   
                 GAGGGATTGCATCGCCCTCGGGCTTTAGTTTCGATAGCCAGCCTCG 
               
               
                   
               
               
                   
                 GTGCGATAAAAAAGGGCTCGACCGATCCAATTCAGCGAGAGCGTCA 
               
               
                   
               
               
                   
                 AGAGTAACTGCCCATTTTGTGAAAGGATTGAAAAAGAGGACGACGC 
               
               
                   
               
               
                   
                 TACGAAAGGACAGCTACCCTCTATCG 
               
            
           
         
       
     
     The disclosure includes targeting any RNA encoding the  Aedes aegypti  alpha mannosidase protein, the sequence of which is as follows. 
     
       
         
           
               
               
            
               
                   
                 &gt;AAEL004389-RB peptide: 
               
               
                   
                 (SEQ ID NO: 3) 
               
               
                   
                 MTVKIFRRGSARCIGLLSAFVTILLCLYY1SMGQPSNTPTTTATSG 
               
               
                   
               
               
                   
                 GSSLHKDAALHQKRLSNLHADPHHGAGSNPNANQSWHSWLRNNLNS 
               
               
                   
               
               
                   
                 INNGGNGKDRPPGLGPEVSDSGGYPDGDGGGGGVGAAAAAVAGSHP 
               
               
                   
               
               
                   
                 PRFSAKWDECVALEETPTDITTGDEYGNFDFQPEWMKTKEYWDKDF 
               
               
                   
               
               
                   
                 ESRYEKLQKDPNRPPLKIVWPHSHNDPGWLKTFVNYFQSDSRQILN 
               
               
                   
               
               
                   
                 LAVTKMPEYNNMSFIWSEISFLQLWWDQAHPTKQRILKKLVKSGRL 
               
               
                   
               
               
                   
                 EITTGGWVMTDEANAHLYAMVDQLIEGHQWVKTNLNVTPKSGWSID 
               
               
                   
               
               
                   
                 PFGHGSTVPYLLAASGFEGTIIQRIHYAWKQWFARHRYGDFLWSPY 
               
               
                   
               
               
                   
                 WRTPSSGLDRKHTLLTHNMPFDIYSIKHSCGPHPFICLNFDFRKIP 
               
               
                   
               
               
                   
                 GEYTEYSIKAQFITPENIESKADLLMEQYSRTASLFPHNVALIPVG 
               
               
                   
               
               
                   
                 DDFRYNKDKEMEQQYTNYKKLIDYINENRNKYKAEISFGTPKDYFN 
               
               
                   
               
               
                   
                 AIKERYDKFPTLKGDFFVYADIFNEGRPAYWSGYFTTRPYYKILSR 
               
               
                   
               
               
                   
                 ELEHNLRSLEILFTLAFNRARQAGNSNAFKIYEKNYEKMILARRNL 
               
               
                   
               
               
                   
                 GLFQHHDAITGTSKANVMRDYALRLFESIQDSVKLQEKTIELLVQK 
               
               
                   
               
               
                   
                 KGTEHNFLIGELERDNFSKLPRKTPLIVTEARSTDFWYNALAQERI 
               
               
                   
               
               
                   
                 EVVLIRTLTPRVKILDPKGNPMNIQINPVWNITETSSYASRKIIPS 
               
               
                   
               
               
                   
                 DKEYEIMFVAKLAPLSLTTFTATYDDEFKPKMATLYCNECQDEKNE 
               
               
                   
               
               
                   
                 IFEIRNKQPGDIQLENFKMRLLFDEQSGFLKSVTKKNMGKQIQCAI 
               
               
                   
               
               
                   
                 KFAAYKSAQFHSGAYLFKTDPEQRNSEKEILEQYNDMTILITSGPL 
               
               
                   
               
               
                   
                 ASDVTAIYGPFLAHTVRIFNSNTVLDNGIFIENDIDFEMPPKNRET 
               
               
                   
               
               
                   
                 ELFMRFVTDIENGASENPEFFSDLNGFQYQKRVKVPSIGIEGNYFP 
               
               
                   
               
               
                   
                 ITSGAFIQDDKMRLTLLTTHAQGAASLEPGQLEVMLDRRTLYDDYR 
               
               
                   
               
               
                   
                 GMGEGVVDSRLTRHRFWVVLENIESHSPPLAENPPGPADEPKPAEF 
               
               
                   
               
               
                   
                 QLPSIFANQLTNGLNYPANLFIVEKYDESNQIELNRAVQLLAAPFP 
               
               
                   
               
               
                   
                 CDLHILNLRTLTEGNLPLFPSSGALLVLHRQGYDCRIGGEEWNYFC 
               
               
                   
               
               
                   
                 NNSSSSVSLSSNSNNYKNVDKYNSRLQLFGGVQIEQITGTSLTGLH 
               
               
                   
               
               
                   
                 PGAPVRSVGDIFLEPMELRTFNLTFVK 
               
            
           
         
       
     
     The following is a representative cDNA sequence of an mRNA encoding  Aedes aegypti  alpha mannosidase protein. 
     
       
         
           
               
            
               
                 {25 
               
               
                 &gt;AAEL004389-RB cdna: protein coding 
               
               
                 (SEQ ID NO: 4) 
               
               
                 CACTACACCGCCTCGCCATTGCATTTTGGACCGTGGAAAGCCGGATCG 
               
               
                   
               
               
                 GGGATCTAATCTTGATGTAGCCGATTACCTCCACACCGTACCCACAAA 
               
               
                   
               
               
                 AAGGATCGCCCGAGTAGAAGAACACTTGAACGTGGCCAGCAGCAGCAG 
               
               
                   
               
               
                 CTTAGACGTCGCCATCATCTATTCGAAGAACAAAAATCCTAGAAAATG 
               
               
                   
               
               
                 TAATTTTCATTCCACCGGCAAGACCAAAGTGAATTAAACTGAACTCCC 
               
               
                   
               
               
                 CCGGATGTGAAGTCCTGTTTTAGCTTGTTGTGTGAGTGTTTGTGTGTC 
               
               
                   
               
               
                 AGAAGACAGAGGAAAAATCATAAAGTGTCACATTCTACCGTGAGTGAA 
               
               
                   
               
               
                 ACGTGAAAGCCGCATCGGCACCCATAAATGAGTGAATATCGCGCGCCG 
               
               
                   
               
               
                 AAAGTTTAGGGTGGAAAATTGCACCGAGTTGGTGGGGTGCTGCTTCCT 
               
               
                   
               
               
                 GTTTATTGCCCCATAATCAAGTGCCGAGGGAGCAGAAGCAGAAAAAAG 
               
               
                   
               
               
                 GTGCCTGCAGCGCCGCAGCATCATGACCGTGAAAATTTTCCGCCGGGG 
               
               
                   
               
               
                 TTCGGCCCGCTGCATAGGGCTCCTGTCGGCTTTCGTAACCATTTTGCT 
               
               
                   
               
               
                 GTGCTTGTACTACATCTCGATGGGACAGCCATCAAACACGCCAACGAC 
               
               
                   
               
               
                 GACCGCCACTTCCGGTGGCTCCTCGCTCCACAAGGATGCTGCCCTGCA 
               
               
                   
               
               
                 CCAGAAACGATTAAGCAACCTTCATGCAGATCCGCACCACGGCGCCGG 
               
               
                   
               
               
                 GAGCAATCCGAATGCAAACCAATCCTGGCACAGTTGGCTGCGGAACAA 
               
               
                   
               
               
                 TCTCAATTCGATCAACAACGGTGGCAACGGCAAGGATCGACCGCCCGG 
               
               
                   
               
               
                 CCTGGGACCGGAAGTGTCCGACAGTGGAGGCTACCCGGATGGTGATGG 
               
               
                   
               
               
                 GGGTGGAGGTGGTGTCGGGGCTGCTGCTGCAGCAGTGGCCGGAAGTCA 
               
               
                   
               
               
                 TCCGCCTCGGTTCAGCGCCAAGTGGGACGAATGTGTCGCACTGGAGGA 
               
               
                   
               
               
                 AACCCCAACCGATATCACCACCGGCGATGAGTATGGAAATTTCGACTT 
               
               
                   
               
               
                 CCAGCCCGAATGGATGAAAACAAAGGAATACTGGGACAAGGACTTCGA 
               
               
                   
               
               
                 GAGCCGTTACGAGAAGCTGCAGAAGGATCCGAACCGACCCCCGTTGAA 
               
               
                   
               
               
                 GATTGTGGTAGTTCCGCACTCCCATAATGACCCCGGGTGGTTGAAGAC 
               
               
                   
               
               
                 CTTCGTCAACTACTTCCAGTCGGATTCGAGGCAGATTCTGAACTTGGC 
               
               
                   
               
               
                 CGTCACTAAGATGCCCGAGTACAACAACATGTCGTTTATATGGAGTGA 
               
               
                   
               
               
                 GATCAGCTTTCTGCAGTTATGGTGGGATCAAGCACATCCCACCAAGCA 
               
               
                   
               
               
                 GAGGATATTGAAAAAGTTGGTGAAATCAGGTCGTTTGGAGATCACTAC 
               
               
                   
               
               
                 TGGAGGCTGGGTCATGACGGATGAAGCGAATGCTCATCTTTATGCGAT 
               
               
                   
               
               
                 GGTTGATCAGCTTATTGAAGGTCATCAATGGGTCAAAACCAATCTGAA 
               
               
                   
               
               
                 CGTAACTCCGAAGAGCGGATGGAGTATAGATCCTTTTGGACATGGTAG 
               
               
                   
               
               
                 TACCGTCCCATACTTGTTAGCAGCAAGTGGTTTTGAAGGAACCATCAT 
               
               
                   
               
               
                 CCAACGGATACACTACGCGTGGAAGCAATGGTTCGCCCGTCATCGATA 
               
               
                   
               
               
                 CGGAGATTTCCTGTGGAGTCCCTACTGGCGAACACCTTCTAGTGGTCT 
               
               
                   
               
               
                 GGATCGAAAGCACACTCTCCTGACTCATAACATGCCCTTCGACATCTA 
               
               
                   
               
               
                 CTCAATCAAACATTCCTGCGGGCCACATCCATTCATCTGCCTTAATTT 
               
               
                   
               
               
                 CGACTTCCGCAAGATTCCTGGCGAGTATACTGAATACTCGATCAAAGC 
               
               
                   
               
               
                 TCAGTTCATCACACCGGAAAACATCGAATCCAAAGCTGACCTTCTCAT 
               
               
                   
               
               
                 GGAGCAATACTCGCGTACTGCTTCCCTGTTCCCTCACAATGTGGCACT 
               
               
                   
               
               
                 GATTCCCGTTGGAGACGATTTCCGTTACAACAAGGATAAAGAAATGGA 
               
               
                   
               
               
                 GCAACAGTACACCAACTACAAGAAGCTGATCGACTATATCAACGAGAA 
               
               
                   
               
               
                 CCGCAACAAGTACAAGGCGGAAATCAGCTTTGGTACTCCGAAGGACTA 
               
               
                   
               
               
                 CTTCAATGCCATCAAGGAACGCTACGATAAATTCCCGACTTTGAAAGG 
               
               
                   
               
               
                 AGACTTTTTCGTCTACGCAGACATCTTCAACGAAGGGCGTCCAGCATA 
               
               
                   
               
               
                 CTGGTCTGGATATTTCACCACCCGACCGTATTACAAGATTCTCAGTCG 
               
               
                   
               
               
                 AGAACTCGAACACAACCTTCGTAGCTTGGAAATTCTGTTCACCTTGGC 
               
               
                   
               
               
                 TTTCAACCGAGCCAGGCAAGCTGGTAATTCCAATGCCTTCAAGATCTA 
               
               
                   
               
               
                 CGAAAAGAACTACGAGAAGATGATCCTTGCTAGGCGGAACCTAGGCCT 
               
               
                   
               
               
                 TTTCCAACATCACGATGCCATCACCGGAACGTCCAAAGCCAATGTGAT 
               
               
                   
               
               
                 GCGAGACTACGCTCTGAGGCTGTTTGAAAGCATCCAAGACTCCGTCAA 
               
               
                   
               
               
                 GCTTCAAGAGAAAACCATAGAACTGCTCGTCCAGAAGAAAGGCACCGA 
               
               
                   
               
               
                 GCACAACTTTCTGATCGGAGAGCTGGAGCGGGATAACTTCAGCAAACT 
               
               
                   
               
               
                 CCCTCGGAAGACTCCTCTGATCGTCACGGAAGCACGGAGTACGGACTT 
               
               
                   
               
               
                 CGTGGTCTACAACGCCCTCGCGCAAGAACGGATAGAAGTCGTTCTGAT 
               
               
                   
               
               
                 CAGAACACTGACCCCGCGCGTTAAAATTCTCGATCCGAAAGGTAACCC 
               
               
                   
               
               
                 AATGAACATACAAATCAACCCGGTGTGGAACATCACGGAAACTTCATC 
               
               
                   
               
               
                 TTACGCATCCCGGAAGATCATTCCCTCGGACAAGGAGTACGAAATCAT 
               
               
                   
               
               
                 GTTTGTGGCGAAGCTGGCACCTCTTTCGCTAACGACCTTTACGGCCAC 
               
               
                   
               
               
                 CTATGACGACGAGTTCAAACCGAAGATGGCAACGCTGTACTGCAACGA 
               
               
                   
               
               
                 GTGCCAAGATGAGAAAAATGAGATATTCGAGATCCGGAACAAACAACC 
               
               
                   
               
               
                 GGGCGACATTCAGCTGGAAAACTTCAAAATGAGGCTGTTGTTTGATGA 
               
               
                   
               
               
                 GCAGAGCGGTTTCTTGAAGTCCGTGACTAAGAAAAACATGGGTAAGCA 
               
               
                   
               
               
                 AATTCAGTGCGCGATCAAGTTTGCCGCGTACAAGAGTGCGCAGTTCCA 
               
               
                   
               
               
                 CTCTGGTGCGTATCTGTTCAAGACGGATCCGGAGCAAAGGAATTCAGA 
               
               
                   
               
               
                 GAAAGAGATACTAGAGCAGTATAATGACATGACAATTCTGATAACTTC 
               
               
                   
               
               
                 CGGCCCACTGGCAAGTGACGTTACAGCAATCTACGGACCATTCCTGGC 
               
               
                   
               
               
                 TCACACCGTGCGGATATTCAACTCCAACACGGTGCTGGATAACGGAAT 
               
               
                   
               
               
                 CTTCATCGAGAATGACATCGACTTTGAGATGCCTCCAAAGAACAGGGA 
               
               
                   
               
               
                 AACAGAACTGTTTATGCGTTTTGTGACAGACATTGAGAATGGGGCTAG 
               
               
                   
               
               
                 CGAAAACCCTGAATTCTTCTCAGATCTTAATGGATTCCAGTATCAGAA 
               
               
                   
               
               
                 GCGAGTGAAGGTCCCATCGATCGGTATCGAGGGCAACTACTTCCCTAT 
               
               
                   
               
               
                 CACCAGCGGGGCATTCATTCAAGATGATAAGATGAGGCTAACTTTGCT 
               
               
                   
               
               
                 CACGACCCACGCTCAAGGCGCTGCCAGCTTGGAACCCGGACAGCTGGA 
               
               
                   
               
               
                 AGTAATGCTCGATAGGCGAACTCTGTACGACGACTATCGTGGTATGGG 
               
               
                   
               
               
                 AGAAGGCGTTGTGGACAGTCGCCTGACCCGACATCGATTCTGGGTTGT 
               
               
                   
               
               
                 TCTAGAGAATATTGAATCCCATTCGCCACCGTTAGCTGAGAACCCTCC 
               
               
                   
               
               
                 GGGGCCAGCTGACGAACCAAAACCCGCCGAATTTCAACTGCCTAGTAT 
               
               
                   
               
               
                 ATTTGCCAATCAGCTCACCAACGGGCTCAACTATCCGGCCAATCTGTT 
               
               
                   
               
               
                 CATCGTGGAAAAGTACGACGAAAGTAACCAGATAGAGCTGAACCGGGC 
               
               
                   
               
               
                 GGTCCAACTGCTGGCCGCTCCGTTCCCCTGTGATCTCCACATTCTGAA 
               
               
                   
               
               
                 TCTCCGAACCCTAACCGAGGGTAACCTGCCCCTGTTTCCGTCGAGCGG 
               
               
                   
               
               
                 AGCTCTGCTGGTTCTACACCGGCAAGGCTACGACTGCCGGATAGGTGG 
               
               
                   
               
               
                 CGAAGAAGTAGTAAATTATTTTTGTAACAATAGTAGTAGTAGCGTAAG 
               
               
                   
               
               
                 TCTTAGTAGTAATAGTAACAATTACAAAAATGTAGATAAGTACAATAG 
               
               
                   
               
               
                 CCGGCTGCAGCTCTTTGGTGGGGTGCAGATCGAACAAATTACCGGCAC 
               
               
                   
               
               
                 GTCGTTAACGGGTTTGCACCCGGGGGCACCGGTGCGTTCCGTGGGGGA 
               
               
                   
               
               
                 CATTTTCCTTGAACCGATGGAACTGCGGACGTTCAACCTGACGTTCGT 
               
               
                   
               
               
                 CAAGTGAGAAGGGGAGAGCGGTGGCGGTGGCGAAGAGACATCAAGCGA 
               
               
                   
               
               
                 AAGGCTTCCACTGGGTTCCTGGTTTTGGATTGGTTTTAGAAAGTTGTT 
               
               
                   
               
               
                 ACAAAGGTGGTCCTTGGATAGTTGGCTGGGAATCAGTTCGGGCTAAAG 
               
               
                   
               
               
                 ATTGGATGTCGGTTTTCTGTGGTCACTTTATGGTGCTTGATTTTAAAA 
               
               
                   
               
               
                 TACGATTGTAAGATTATCTTTCTTTCATGCCGATACCAGATCCGAGTA 
               
               
                   
               
               
                 GCTGCATTCTTTTAACGTGAAGGGTCGGCTTGACATAATTTATCCAGG 
               
               
                   
               
               
                 CATTTGCCCAGAAGATTATTTTTTATGGTGCTATGACCTGCGCCACAA 
               
               
                   
               
               
                 TGTTACGACAACAAACTCGATTGCCTCTAGTTTCTTGATCGTCCCAAA 
               
               
                   
               
               
                 CTTCCAAGGTCCTGCTCTACTTGGTCATGCCACCTAAGTCGGAGCGCC 
               
               
                   
               
               
                 CTTCCCCGGCCGGATTCGAGGTGAACTCCATTTTTGGAGGATTTATCA 
               
               
                   
               
               
                 CAATTACCCAGCCCATCGTACTCTTTCAGCATTGAAGACTTTCTAGAT 
               
               
                   
               
               
                 ATATCAAGTTCACCGTAGAGTTGCGCAAGCTATTGGTTCATACGTTTG 
               
               
                   
               
               
                 CTGGCATGAATTTCACTCAAAAGGCTTCGGTGTGATGCAAACGCAATA 
               
               
                   
               
               
                 AATTTTTACTCAGACGTTGTTGCAAGGGTTTGAACGGCTTGCTCATTG 
               
               
                   
               
               
                 GTTGATTCAAACACAGAGTGTAATAAGAAAAACTCAGCAATCACAGAA 
               
               
                   
               
               
                 AAAGAAAACCGTCTTCGTGAGTCTCGTCTCAGTTCTTAAGCGTTTCCT 
               
               
                   
               
               
                 ACGCATCGAGATAGGCGATTATTATTCTTGTCAGTAGCAAACCAGCCC 
               
               
                   
               
               
                 TTAAGCCAGATTTGTTGCTGAAAAGGAACCGCTAAAGACGTTAATCGC 
               
               
                   
               
               
                 CGACTTCTTGCCGAAATTTCCTATAGTGAGTTCCGTTTGAACTGACAT 
               
               
                   
               
               
                 TTCTTTTCAACTGCGTACTGCGATCAGAAAAAGGGCTTTCTTGATCGA 
               
               
                   
               
               
                 TTTGTTACAGGCATTTCCCATAGATATTTAAGCAAATCAGGCTTTAGC 
               
               
                   
               
               
                 CTGTTTTACTGCTATCTGTAACGTTTTTCCGATGGTTCATTTGATGTG 
               
               
                   
               
               
                 GAAATTAGTTTAACGGGTTCAATCGCCCTCGTACCTATCCCTTGGGTT 
               
               
                   
               
               
                 CAATATATGCAAAGAAGTATTAGTGACATGACATGATAGACAGTCATA 
               
               
                   
               
               
                 ATCAACGAGGGACGATGTATACATCCTATAGGAATACGAATTGCAGAG 
               
               
                   
               
               
                 ATTGTGACGGGAATAAGTGCGGTGATCCTTCATTAAATCCGAAATGCG 
               
               
                   
               
               
                 AAAACTGCAGATATAGTGCGCTTATCGGACTTGTAGTTCAGAACCATT 
               
               
                   
               
               
                 GCATTTGAATTTTTTGCTCTTATTCATTTTACTGTTACTGTGCATAAA 
               
               
                   
               
               
                 CTAAAGATGCTACAAATAACAGATATCTAAGTCCAAATTGAAAACTGG 
               
               
                   
               
               
                 CAAGAATGTTAACCACAAACGATGATTTAACGGCCTTTCAAACTGGCA 
               
               
                   
               
               
                 ACACGTGTAGCGTCGCTGATATCATGGTGAAAATGAAACATTTCAGGA 
               
               
                   
               
               
                 TGTTCTTAATCACTACCAATTAACACAACATGCTGATCCACGTTGATA 
               
               
                   
               
               
                 TGTGGGTCAAGTGATATCACCATAGTTGGGTTTGAATGCTTATACAAC 
               
               
                   
               
               
                 TTTTGAGAAGGTTTTTGTTCCAACATCGATAACTTTTCCTGTGCTTGA 
               
               
                   
               
               
                 ACTTAGATACTAAGACAGCAAATGTCAAAAATATGAGTCACTTGTGGA 
               
               
                   
               
               
                 AATGTCTTCTAAGTCATCGCCATTTGTCTCTCATTGCTTCTACAGAAA 
               
               
                   
               
               
                 TTGAATTCGATCATCAAGACCAAGCTATCAAAACCAGATACGGAATCA 
               
               
                   
               
               
                 AACATAAGAAAATACAAACGACATCACTCTCTTCCAAGTCTATCCGAT 
               
               
                   
               
               
                 ATGTGAATCTCACAAATATTGATCTGAACTAGGGCAACAATTTTTCGA 
               
               
                   
               
               
                 ATATGGTTTTAAGTTATTTTATTGAAAACAAAAACAGCAGCTGAATCA 
               
               
                   
               
               
                 TTTCACACGACATAGACGAATCTAAGCAGAACCCCTCAATGTAATCAT 
               
               
                   
               
               
                 TCCACTTTTTAAGAGAAGAAAAAACAAGAAATCTGGTCCAGTATATAT 
               
               
                   
               
               
                 TGTTAGATTTATTATTACTGAACAATGCAAAAAATCCCCACAATTTAA 
               
               
                   
               
               
                 TTTTATGTTCGAAACAAAAAGTGCATGTACCATTCAAGTTGGAAAGAA 
               
               
                   
               
               
                 ACCTCTTTGCTCTGTATTGTACATACCAAAATCTAGCCCGTAGAATCT 
               
               
                   
               
               
                 CCTTATCTACATACTTTTGTGATTATAGTGTTAGTGATACGTTTCTTC 
               
               
                   
               
               
                 CCTGATTCTTCGATTTCTGTTGAAAACTTCCATTCATTTCCATCCTGG 
               
               
                   
               
               
                 GTCCAATTTAAATACCTCTATCCTTCACAAACGATTTGTTCTTTCAAT 
               
               
                   
               
               
                 CACAGCTATGATATTTAAAGTTTTAACCACCGTCCAGCAGGATTAGTT 
               
               
                   
               
               
                 GTTGTCACATAAAATTCCCTACTTTTATTAGTTACTATTTATTAATTA 
               
               
                   
               
               
                 AATCTAGGATACCCCTAAAATAGTGAGCGTGCGGACGAGCAGCTAATC 
               
               
                   
               
               
                 CCAAATGAGCCCCTGATATAAACTTGTTTGATATTATCCTACATAATA 
               
               
                   
               
               
                 ACAACTACGATCAAAAGCGATTTTACAAAGAAAAAAAAAGCAAAGCAC 
               
               
                   
               
               
                 CTACAAGGTAATGCAAAAGAGCAAAACAAAAAAAACTAGCAAAAGATA 
               
               
                   
               
               
                 GTAAGTAAGCAAATCATCGCGTATGTTTAGAAAAAGGATTAGTTGTTT 
               
               
                   
               
               
                 TATAAAAGGAAATGAACCTACTGCAGCTAGAAATTAAATTTTAAAGGA 
               
               
                   
               
               
                 AATAACGGATCAACCGAGCAGGAATGAACAACCATATTCTTAAACTAG 
               
               
                   
               
               
                 AAGAGAAAGAATTTAACTATTTTATTGTTTATTGTCCCGTCGTTGAAT 
               
               
                   
               
               
                 CTCCGTTTATTTTACATTGATGTCTAAAGCGTCTGTCAGAAAATGTGT 
               
               
                   
               
               
                 GACCAGTTGTAGTAAAATTTGTTTTTAACAGTAAATTAACCATTTGTG 
               
               
                   
               
               
                 CAGCTCGGAAGTTTACAGTTGATTTCATTGCAAAAAGTAATCATTACA 
               
               
                   
               
               
                 TTTTTTGTTGCAAAATATGTATTGATAGAACAAA 
               
            
           
         
       
     
     In embodiments, the disclosure provides use of an RNA-agent to inhibit translation of an mRNA, or to degrade the mRNA encoding a protein described herein. In embodiments, the RNAi agent is not completely identical in length and sequence to an mRNA expressed by a mosquito. In embodiments, the RNAi agent comprises a segment of RNA that is targeted to an mRNA produced by a mosquito. 
     In embodiments, any RNA sequences or derivatives thereof described herein can be adapted for use as an RNAi agent, and such sequences may be modified in a variety of ways. In embodiments, the RNAi agent is used as an shRNA. The disclosure includes direct shRNA administration, and administering a vector that encodes the shRNA. In embodiments, the RNAi agent comprises a microRNA, and thus comprises direct administration of a microRNA, and administration of a vector that encodes the microRNA. The term “microRNA” can be used interchangeably with “miR,” or “miRNA” to refer to, for example, an unprocessed or processed RNA transcript from an engineered miRNA gene. The unprocessed miRNA gene transcript is also called a “miRNA precursor,” and typically comprises an RNA transcript of about 70-100 nucleotides in length. The miRNA precursor can be processed by digestion with an RNAse (for example, Dicer, Argonaut, or RNAse III) into an active 19-25 nucleotide RNA molecule, non-limiting examples of which are described above. This active 19-25 nucleotide RNA molecule is also called the “processed” miRNA gene transcript or “mature” miRNA. Any of these forms of microRNA can be adapted for use in embodiments of this disclosure. Further, in certain embodiments, the RNAi agent may be provided as a synthetic agent, such as a microRNA mimic, short interfering RNA (siRNA), a RNA interference (RNAi) molecule, double-stranded RNA (dsRNA), short hairpin RNA (shRNA), primary miRNAs (pri-miRNAs), small nucleolar RNAs (snoRNAs), a molecule capable of sequence-specific post-transcriptional gene silencing of miRNA, or any combination thereof, where the RNAi agent inhibits expression of the protein. Inhibition of the expression may therefore be achieved by inhibiting translation, transcription, and/or by mRNA degradation. In embodiments, a double-stranded (ds) RNA may be administered to a mosquito, and non-limiting examples of such dsRNA and their efficacy in reducing viral load are demonstrated in the examples. 
     The disclosure includes, without limitation, modified bacteria that express an RNAi agent described herein, and includes modified mosquitoes that express any such RNAi agent. The disclosure includes isolated RNAi agents, and any type of vector, including but not limited to viral vectors and plasmids that encode and are capable of expressing the RNAi agents in bacterial, and/or in insect cells, particularly mosquito cells. In embodiments, the disclosure provides modified viruses, such as viral particles, such as bacteriophages, that encode the RNAi agent, as well as phagemids that encode the RNAi agent. In embodiments, bacteriophages that are modified to comprise a genome that encodes a RNAi agent described herein are provided. 
     In certain implementations, an RNAi agent is expressed by a modified mosquito, or is introduced into a mosquito. In embodiments, the modified mosquito is, absent a modification described herein, susceptible of being a vector for one or more viruses, including but not limited to viruses that are DENV, ZIKV, CHIKV, or any combination thereof. In embodiments, a mosquito modified according to this disclosure is an  Aedes aegypti  mosquito. Thus, the disclosure provides for limiting, reducing, replacing, or eradicating susceptible mosquitoes from a mosquito population using modified mosquitoes that express an RNAi agent described herein. In embodiments, use of an RNAi agent as described herein reduces viral load in a mosquito. In non-limiting embodiments, use of an RNAi agent as described herein results in a reduction of DENV viral load in a mosquito. In embodiments, a reduction in viral load occurs in mosquitoes that are free of  Wolbachia  bacteria. In embodiments, a reduction in viral load occurs in mosquitoes that are infected with  Wolbachia.  Determination of viral load can be determined according to techniques that are well known in the art, including but not limited to PCR-based methods. 
     A mosquito modified to express or comprise an RNAi agent described herein can be modified as such in any suitable way, such as by introducing an episomal element that encodes the RNAi agent into mosquito cells so that the an RNAi agent is expressed. Expression of the RNAi agent may be transient or constitutive, and the expression of an RNAi agent may be inducible, such as being inducible by one or more virally encoded transcription factors. In embodiments, a mosquito chromosome is modified at a larval stage. In embodiments, one or more mosquito totipotent, pluripotent, or multipotent stem cells are modified. In embodiments, only one sex of mosquitoes in a mosquito population is modified. In embodiments, only female mosquitoes are modified. In embodiments, a mosquito chromosome is edited such that it can express an RNAi agent, wherein the modification is made using any suitable chromosome editing technique, including but not limited to CRISPR-based approaches as well as Talens and HEGs. The RNAi agent can be expressed using any promoter that can function in mosquito cells, including but not limited to a recombinantly-introduced promoter that is operably linked to the RNAi agent coding sequence. Additionally an infectious viral agent including but not limited to baculovirus could be used to introduce the gene into the mosquito genome. 
     In embodiments, the disclosure comprises introducing into mosquitoes an RNAi agent, and optionally a polynucleotide or other element that confers resistance to, for example, a pesticide, and thus comprises a resistance element. Accordingly, borrowing from previous approaches in the agricultural industry to, for example, control the growth of weeds, and/or to promote survival and/or reproduction of plants or the production of viable plant seeds that are resistant to a pest or pesticide or herbicide, the disclosure provides mosquitoes that are resistant to a selection agent via concomitant expression of the RNAi agent and at least one resistant agent that confers resistance to a selection agent. Using this approach, mosquitoes that are not resistant to harbouring viruses, due to a lack of expression of the RNAi agent and the resistance element, can be reduced or eliminated from a mosquito population. In embodiments, the disclosure comprises releasing modified mosquitoes into an environment. In embodiments, releasing the mosquitoes comprises at least a part of performing a gene drive to generate a population of mosquitoes that express or comprise an administered RNAi agent. 
     In certain embodiments, an RNAi agent is expressed by a modified bacteria. The bacteria may or may not be modified  Wolbachia,  and as such the disclosure includes any modified bacteria that are capable of infecting, and/or living symbiotically or commensally within mosquitoes. In embodiments, the modified bacteria is a bacteria that is adapted to live intracellularly in mosquito cells. Generating bacteria that express an RNAi agent can be performed using any of a wide variety of well-known techniques, such as by introducing a plasmid into the bacteria that encodes the RNAi agent, such that the RNAi agent is expressed by the bacteria. The plasmid may have any feature, such as a selectable marker, or any other component that facilitates its persistence in a bacteria population. The disclosure includes all bacteria, vectors and plasmids, plasmid cloning intermediates, primers, PCR amplifications, PCR amplicons, restriction enzyme digests, which are or could be generated while constructing final vectors or plasmids described herein. A range of bacterial species could be used, including any that naturally infect the mosquito gut and that could be grown and genetically modified. 
     It will be recognized from the foregoing that the disclosure includes methods for controlling viruses described herein by introducing into mosquitoes any RNAi agent, and further includes introducing an RNAi agent as a component of a pharmaceutical formulation, or as a component of a substance that is consumed by mosquitoes, or by introducing modified bacteria into the mosquitoes, or by introducing a DNA polynucleotide that is capable of expressing the RNAi agent into mosquitoes. 
     In embodiments, any RNAi agent or derivatives thereof described herein are used as a microRNA. The term “microRNA” can be used interchangeably with “miR,” or “miRNA” to refer to, for example, an unprocessed or processed RNA transcript from an engineered miRNA gene. The unprocessed miRNA gene transcript is also called a “miRNA precursor,” and typically comprises an RNA transcript of about 70-100 nucleotides in length. The miRNA precursor can be processed by digestion with an RNAse (for example, Dicer, Argonaut, or RNAse III) into an active 19-25 nucleotide RNA molecule, non-limiting examples of which are described above. This active 19-25 nucleotide RNA molecule is also called the “processed” miRNA gene transcript or “mature” miRNA. Any of these forms of microRNA can be adapted for use in embodiments of this disclosure. Further, in certain embodiments, the RNAi agent may be provided as a synthetic agent, such as a microRNA mimic, short interfering RNA (siRNA), a RNA interference (RNAi) molecule, double-stranded RNA (dsRNA), short hairpin RNA (shRNA), primary miRNAs (pri-miRNAs), small nucleolar RNAs (snoRNAs), a molecule capable of sequence-specific post-transcriptional gene silencing of miRNA, or any combination thereof, where the RNAi agent inhibits expression of the protein. Inhibition of the expression may therefore be achieved by inhibiting translation, transcription, and/or by mRNA degradation. 
     In embodiments, the RNAi agent may be modified to improve its efficacy, such as by being resistant to nuclease digestion. In embodiments, the RNAi agent polynucleotides which comprise modified ribonucleotides or deoxyribonucleotide, and thus include RNA/DNA hybrids. In non-limiting examples, modified ribonucleotides may comprise methylations and/or substitutions of the 2′ position of the ribose moiety with an —O— lower alkyl group containing 1-6 saturated or unsaturasted carbon atoms, or with an —O-aryl group having 2-6 carbon atoms, wherein such alkyl or aryl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or with a hydroxy, an amino or a halo group. In embodiments modified nucleotides comprise methyl-cytidine and/or pseudo-uridine. The nucleotides may be linked by phosphodiester linkages or by a synthetic linkage, i.e., a linkage other than a phosphodiester linkage. Examples of inter-nucleoside linkages in the polynucleotide agents that can be used in the disclosure include, but are not limited to, phosphodiester, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphate ester, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, morpholino, phosphate triester, acetamidate, carboxymethyl ester, or combinations thereof. 
     In non-limiting demonstrations, we used dsRNAs as RNAi agents. This approach achieved reduction in viral load in mosquitoes, as follows. 
     We used dsRNA targeted to  Aedes aegypti  alpha-mannosidase 2 mRNA as an RNAi agent. The dsRNA comprised the following RNA sequence, along with its complementary strand: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 5) 
               
               
                 GGGAUAACUUCAGCAAACUCCCUCGGAAGACUCCUCUGAUCGUCACGGA 
               
               
                   
               
               
                 AGCACGGAGUACGGACUUCGUGGUCUACAACGCCCUCGCGCAAGAACGG 
               
               
                   
               
               
                 AUAGAAGUCGUUCUGAUCAGAACACUGACCCCGCGCGUUAAAAUUCUCG 
               
               
                   
               
               
                 AUCCGAAAGGUAACCCAAUGAACAUACAAAUCAACCCGGUGUGGAACAU 
               
               
                   
               
               
                 CACGGAAACUUCAUCUUACGCAUCCCGGAAGAUCAUUCCCUCGGACAAG 
               
               
                   
               
               
                 GAGUACGAAAUCAUGUUUGUGGCGAAGCUGGCACCUCUUUCGCUAACGA 
               
               
                   
               
               
                 CCUUUACGGCCACCUAUGACGACGAGUUCAAACCGAAGAUGGCAACGCU 
               
               
                   
               
               
                 GUACUGCAACGAGUGCCAAGAUGAGAAAAAUGAGAUAUUCGAGAUCCGG 
               
               
                   
               
               
                 AACAAACAACCGGGCGACAUUCAGCUGGAAAACUUCAAAAUGAGGCUGU 
               
               
                   
               
               
                 UGUUUGAUGAGCAGAGCGGUUUCUUGAAGUCCGUGACUAAGAAAAACAU 
               
               
                   
               
               
                 GGGUAAGCAAAUUCAGUGCGCGAUCAAGUUUGCCGCGUACAAGAGUGCG 
               
               
                   
               
               
                 CAGUUCCACUCUGGUGCGUAUCUGUUCAAGACGGAUCCGGAGCAAAGGA 
               
               
                   
               
               
                 AUUCAGAGAAAGAGAUACUAGAGCAGUAUAAUGACAUGACAAUUCUGAU 
               
               
                   
               
               
                 AACUUCCGGCCC 
               
            
           
         
       
     
     We used dsRNA targeted to  Aedes aegypti  cadherin mRNA as an RNAi. The dsRNA comprised the following RNA sequence, along with its complementary strand: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 6) 
               
               
                 GGGACAUCCGCAUCGGUAAAAUCCAGGCCUAUUACGACACACCCGACC 
               
               
                   
               
               
                 CGAAAAUCUACUACUACAUGAUGCUCGGCAACGAGGAUGGAGCGUUCU 
               
               
                   
               
               
                 ACGUGGACAAAACCACCGGCGAUAUCUACACCAACAAAACGCUGGACC 
               
               
                   
               
               
                 GCGAGGAAGCGGAUGUCUACGCUCUCUAUAUCAAAGCCAGCAAGAAAC 
               
               
                   
               
               
                 AAGACCUGCUGAUCACUGAGCGCGAUCGGAUGAUGAUGUCGACCAAAA 
               
               
                   
               
               
                 AGCUGGAACGCGAUAGCACGGUUGCGAAGGUCUGGAUCACAGUCCUCG 
               
               
                   
               
               
                 AUGUCAACGACAAUCCCCCGGUCUUUAAACAGGACGUUUACUACGCUG 
               
               
                   
               
               
                 GCGUAAGCUCCAAGGCUGCCAUCAACGAAUUGGUGACAAUUGUCAAUG 
               
               
                   
               
               
                 CGACCGAUCGAGAUCUGGGCGUGAACUCUACCAUGGAACUGUUCAUCA 
               
               
                   
               
               
                 GCGGGUCUUAUCUUUACAAAUACGGAGCUACGAAGACAACUGGUAGCA 
               
               
                   
               
               
                 UAGUUCCAAGUCCGUUCACUAUUUCCAAGGACGGUCGUAUAACUACCG 
               
               
                   
               
               
                 CAAACUACAUGGCCGAAUAUAACCAGGACCGUUUCAUUCUGGACAUUG 
               
               
                   
               
               
                 UAGCAAAAGAGGUGGAAUCUCCUGAGCGAGUUGCCACCACCAAAGUCU 
               
               
                   
               
               
                 ACGUCUGGAUCUUCAAUCCAGAACAACUAGUGCGUGUGAUCCUGUCGA 
               
               
                   
               
               
                 GGCCACCC 
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                 TABLE B 
               
               
                   
               
               
                   
                   
                   
                 Primer 
                 dsRNA 
               
               
                 dsRNA 
                 Vector- 
                   
                 Sequence 
                 amplicon 
               
               
                 name 
                 base 
                 Primer 
                 (5′-3′) 
                 size 
               
               
                   
               
             
            
               
                 dsAlpha2 
                 AAEL0 
                 Forward 
                 taatacgac 
                 649 bp 
               
               
                   
                 04389 
                   
                 tcactataG 
                   
               
               
                   
                   
                   
                 GGATAACTT 
                   
               
               
                   
                   
                   
                 CAGCAAACT 
                   
               
               
                   
                   
                   
                 CC 
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 7) 
                   
               
               
                   
                   
                 Reverse 
                 taatacgact 
                   
               
               
                   
                   
                   
                 cactataGGG 
                   
               
               
                   
                   
                   
                 CCGGAAGTTA 
                   
               
               
                   
                   
                   
                 TCAGAAT 
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 8) 
                   
               
               
                   
               
               
                 dsCadherin 
                 AAEL02 
                 Forward 
                 taatacgact 
                 680 bp 
               
               
                   
                 3845-RA 
                   
                 cactataGGG 
                   
               
               
                   
                   
                   
                 ACATCCGCAT 
                   
               
               
                   
                   
                   
                 CGGTA 
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 9) 
                   
               
               
                   
                   
                 Reverse 
                 taatacgact 
                   
               
               
                   
                   
                   
                 cactataGGG 
                   
               
               
                   
                   
                   
                 TGGCCTCGAC 
                   
               
               
                   
                   
                   
                 AGGAT 
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 10) 
               
               
                   
               
            
           
         
       
     
     In Table B, Lowercase letters=T7 promoter tag. Table B provides the primers that were used to produce the dsRNA constructs described above: 
     In more detail, RNA silencing of  Aedes aegypti  alpha-mannosidase 2 and  Aedes aegypti  Cadherin87A gene expression was performed using the dsRNA constructs described above. The dsRNAs were synthesized using standard techniques using the primers described above, and injected into  Aedes aegypti  mosquitoes, followed by viral challenge via a blood meal. Control dsRNA constructs were targeted to green fluorescent protein mRNA, which has no known homolog in mosquitoes. Introduction of the dsRNA was performed by adapting known techniques, such as those described in Pan X, et al., Proc Natl Acad Sci U S A. 2012 Jan. 3; 109(1):E23-31, the disclosure of which is incorporated herein by reference. Subsequent to viral challenge by dengue virus serotype 2 via a blood meal, mosquitoes were sacrificed and viral load was assessed by PCR using known approaches. PCR was performed on tissue from scarified mosquitoes, which were sacrificed at the indicated time point post infection (DPI). Results in  FIG. 11  show reduction in dengue virus load following RNA silencing of the cadherin gene in the mosquito carcass. Specifically, a reduction in dengue virus load following RNA silencing of the cadherin gene in the mosquito carcass 14 days post infection of  Wolbachia -infected mosquitoes was achieved. These differences demonstrate that the expression of the cadherin gene is assisting with control of dengue virus in the presence of  Wolbachia  infection. Manipulation of Cadherin expression as described herein is therefore expected to strengthen  Wolbachia -mediated blocking of viruses in mosquitoes. 
     For alpha mannosidase, we see a reduction in dengue virus load following RNA silencing of the same gene relative to GFP controls in mosquitoes not infected with  Wolbachia  at 10 days post infection in the midgut ( FIG. 12 ). These data indicate that the targeting of alpha-mannosidase expression by as described herein may be expected to render wild type mosquitoes less able to replicate dengue virus in their midguts, thereby reducing transmission rates. 
     With respect to  Wolbachia,  it is known in the art as an alpha-proteobacterium that lives within the cells of approximately 40% of all insect species 7  and is transmitted from female insects to their offspring 8 . This bacterium has two traits that have made it a candidate for the biological control of mosquito-borne viruses: first,  Wolbachia  can spread rapidly through populations of insects via reproductively manipulating the hose; and second,  Wolbachia  has been found to limit viral replication in insects, a phenotype that is referred to as pathogen ‘blocking’ 9-11 . Although not naturally found in  A. aegypti,  the bacterium was stably introduced into the species via microinjection over a decade ago 12 .  Wolbachia&#39;s  ability to reduce the transmission potential of DENV, ZIKV and CHIKV 9,13-17  has formed the basis of trial releases into mosquito populations throughout the tropics 18 .  Wolbachia  has successfully spread through  A. aegypti  field populations and remained at high frequencies 18-23 . The impact of these releases on the incidence of human disease is still unknown, but is being assessed 18 . 
     The longevity of  Wolbachia&#39;s  use as a disease control agent will depend on the stability of its blocking phenotype over time 24 . For example, the Myxoma virus (MYXV) used against European and Australian rabbits, illustrates how evolutionary change in either the agent or the target can lead to reduced effectiveness. In the years after releases, the virus evolved into less virulent forms 25  and the host evolved resistance  26 . In its native South American rabbits, MYXV was less virulent than in the naive target populations, suggesting that the evolution of lower virulence was adaptive. Similarly, there is concern that pathogen blocking may evolve to be less effective in the recently infected  A. aegypti  over time, since  Wolbachia  densities and viral blocking tend to be lower in natively infected hosts 1,27-29 . 
     Predicting the long-term stability of  Wolbachia -mediated blocking is particularly challenging because we do not understand the underlying genetic mechanism. There is some evidence that  Wolbachia  may compete with viruses for host resources30,31, induce a heightened basal immune response in the host or manipulate host gene expression via the production of small RNAs 32 . None of these effects, however, fully explain the blocking phenotype 24 . Nevertheless, it is most widely observed that stronger blocking is associated with higher  Wolbachia  loads and broader tissue distributions 24,33-35 . While fitness costs for  Wolbachia  infection tend to be mild when measured in controlled laboratory environments, there is evidence that they increase with increasing  Wolbachia  density 19 . Thus, it has been predicted that selection could favour reduced  Wolbachia  density and so blocking 24 . 
     In the present disclosure, we used artificial selection to dissect genetic variation in the strength of  Wolbachia -mediated DENV blocking in  A. aegypti  hosts and its effects on host fitness. Without intending to be constrained by any particular theory, our aim was to determine how  Wolbachia -based biocontrol could persist by measuring: 1) genetic variation for blocking; 2) the genetic basis for blocking; and 3) how blocking may be maintained by natural selection 24,27 . We selected for high and low DENV blocking alongside a control treatment where mosquitoes were selected at random ( FIG. 1 ). Each treatment was performed on 3 independent lines and all lines were initiated from the same ancestral population of  A. aegypti  mosquitoes from Queensland, Australia that were infected with the wMel strain of  Wolbachia.    
     As a result of these tests, and others that are described more fully by the description and figures presented below, the present disclosure provides the aforementioned approaches to use of existing  Wolbachia  strains and/or RNAi agents to controlling viruses, as well as other modified bacteria and approaches that are described above. 
     From the analysis described herein, we found significant genetic variation for  Wolbachia -mediated DENV blocking, resulting in a rapid response to selection. Moreover, the magnitude of blocking was correlated with  Wolbachia  density. We reveal that genetic variation in both  A. aegypti  and  Wolbachia  affected blocking strength and that this was strongly associated with mutations in  A. aegypti  genes involved in cell-to-cell adhesion and  Wolbachia  genes involved in translation and bacterial cell wall biosynthesis. Finally, we discovered that populations with high viral blocking had faster population growth, indicating the potential for  Wolbachia -mediated DENV blocking to be maintained by natural selection within  A. aegypti.    
     The following examples are intended to illustrate, but not limit the present disclosure. 
     Variation in Blocking Strength 
     To determine the degree of genetic variation for  Wolbachia -mediated DENV blocking in  A. aegypti  we selected upon DENV load for 4 mosquito generations. We found that  Wolbachia -mediated DENV blocking evolves rapidly, with significant divergence in phenotypes occurring after just 4 generations ( FIG. 2 a    Mixed effects model: Treatment: Chisq=9.68, df=7, P=0.0079). DENV loads were significantly higher in the lines selected for low blocking relative to lines selected for high blocking (Post-hoc Tukey comparisons: L-H: 0.0021). The Low blocking lines also had significantly higher DENV loads than the Random blocking lines and there was no significant difference between the Random and High blocking lines (Post-hoc Tukey comparisons: L-R: 0.0006, R-H: 0.94). When we compared the treatments over time, we detected a significant interaction between Treatment and Generation ( FIG. 2 b    Mixed effects model. Treatment: Chisq=6.73, df=7, P=0.03; Generation: Chisq=0.1, df=9, P=0.95; Treatment*Generation: Chisq=20.2, df=13, P=0.00046). 
     By removing  Wolbachia  from each of the evolved lines with the antibiotic tetracycline 36,37  (see  FIG. 7  for confirmation of  Wolbachia  removal), we found no significant difference between mosquito resistance to DENV ( FIG. 2 c    Wolb− Mixed effects model. Treatment: Chisq=1.25, df=7, P=0.53, FDR-corrected P-value=0.69), confirming that the observed variation in DENV load is a result of  Wolbachia -mediated DENV blocking ( FIG. 2 c    Wolb+ Mixed effects model. Treatment: Chisq=12.9, df=7, P=0.0016, FDR-corrected P-value=0.0032. Post-hoc Tukey comparisons: H-R: P=0.95. R-L: P&lt;1e−05. H-L: P=2.14e−05). On average, the Low blocking lines were 40% less effective at reducing DENV load than both the High and Random blocking lines. 
       Wolbachia  Density 
     Several studies have found a heritable basis for  Wolbachia  density that also correlates with the strength of viral blocking 24,33-35 . Here we examined if  Wolbachia  density changed in response to selection and played a role in the observed divergence in blocking strength. To increase sensitivity, we removed the ovaries from each mosquito and analysed them separately as they are known to contain disproportionately high densities of  Wolbachia   38 . In agreement with the literature, we identify a negative correlation between  Wolbachia  density and DENV load across the evolved lines within the bodies of mosquitoes ( FIG. 3  Linear regression.  Wolbachia  density: t=−2.9, df=7, P=0.02*, R 2 =0.55). We found this relationship only in the bodies of mosquitoes, not in the ovaries ( FIG. 8 , Linear regression.  Wolbachia  density: t=-0.56, df=7, P=0.59, R 2 =0.042). 
     Genetic Basis of Variation 
     To understand the genetic basis underlying the phenotypic divergence observed in blocking, we sequenced pools of 90 individual mosquitoes from the ancestral populations and from each evolved line at generation 4 and looked for single nucleotide polymorphisms (SNPs) that were significantly differentiated between treatments by performing pairwise whole genome Cochran-Mantel-Haenszel (CMH) tests. Our threshold for significance was set as the smallest P-value from comparing the Random blocking populations with the ancestral population. This is based upon the assumption that differences between these populations are due to drift and so are false positives. Based upon this threshold, we found significantly differentiated SNPs in both  A. aegypti  and  Wolbachia  genomes when we compared the lines from the High blocking and Low blocking treatments, suggesting that both organisms played a role in determining the phenotypic extremes of  Wolbachia -mediated DENV blocking ( FIG. 4  and  FIG. 5 ). 
     In  A. aegypti  there were approximately ˜60 genes with significant SNPs differentiating the High and Low lines (Table 1). There was a particularly significant peak on chromosome 1 (labelled as region A in  FIG. 4 a   ), containing SNPs in two neighbouring genes. One of these genes is predicted to encode the cadherin-87A protein (AAEL023845), a glycoprotein that is involved in cell-to-cell adhesion 39 . We found 84 SNPs within this gene that have P-values smaller than the significance threshold. The other gene encodes alpha-mannisodase 2a (AAEL004389), an enzyme in the N-glycan biosynthesis pathway that plays a role in the functioning of cadherin 40,41 . We found 15 SNPs within this genes that have P-values smaller than the significance threshold. By performing CMH tests between the other evolved populations and the ancestral population, our data indicate that mutations associated with strong blocking in these genes that were frequent in the High blocking populations were also frequent in the ancestral and Random blocking populations. This is because, the peak of differentiation in these genes is not present in comparisons between the High, Random and ancestral populations. Consistent with this, SNPs in region A were also highly differentiated when we compare the Low blocking populations with the Random blocking populations ( FIG. 4 c   ) and the ancestral population ( FIG. 4 e   ). This therefore indicates that there could be a fitness advantage of stronger blockers, such that this phenotype is at a high frequency in the base population prior to directional selection and is maintained in the Random treatment in the absence of directional selection. 
     We find two main regions where the High and Random blocking populations differ, denoted B and C ( FIG. 4 ). Although SNPs in these regions show some differentiation with the Low blocking populations, they are likely to be less critical for blocking strength since both High and Random blocking populations show strong blocking and differ in this region. 
     Far fewer differentiated SNPs were identified in the  Wolbachia  genome overall (see  FIG. 5 ), consistent with its recent introduction into  A. aegypti  by microinjection 21 . By comparing lines from the High blocking and Low blocking treatments ( FIG. 5 a   ) we found differentiated SNPs within 14 genes that can be grouped by function (Table 1). These include: 1) five genes involved in mRNA translation; 2) four genes involved in the biosynthesis of the bacterial cell wall component, peptidoglycan; 3) three genes involved in stress response; and 4) two genes involved in changing DNA topology. Interestingly, each of the 5 genes involved in mRNA translation are either tRNAs or are translational machinery that bind tRNAs, suggesting some functional role of tRNAs. It has been shown that bacterial tRNAs can be reduced into small noncoding RNAs that can control gene expression 42,43 . We hypothesised that  Wolbachia  could be producing tRNA-derived small noncoding RNAs that alter host gene expression. We performed a BLAST search of the tRNA-Ile gene against the  A. aegypti  genome and found 95% identity across 20nt near the 3′ end of the tRNA with the host gene DnaJ (AAEL005305). This gene encodes a heat shock protein in the 40 family (Hsp40) that is a co-chaperone in the Hsp70 chaperone function. 
     Implications of Variation on  A. aegypti  Fitness 
     To understand how the observed genetic variation could shape the evolution of  Wolbachia -mediated viral blocking and so the stability and success of  Wolbachia  as a biological control strategy, we investigated the impact of the different genotypes on mosquito fitness. More specifically, we calculated the population growth rate (r) of the mosquitoes in the absence of DENV infection to estimate how fitness varies with blocking strength. We did this by measuring: median time to pupation, adult sex ratio, female adult survival, the number of eggs laid per female over 3 bloodmeals and the rate of egg laying. We combined these data to construct Leslie matrix models to gain an estimate of population growth rate (r) for each line 44 . To check the robustness of our findings we tested models across two values of larval survival to adulthood that were chosen to represent the low and high extremes (43% and 92%, respectively) of the range observed experimentally. 
     We found a significant negative correlation between DENV load per mosquito and  A. aegypti  population growth rate (r) across both low larval survival ( FIG. 6  Mixed effects regression controlling for hatch order. Log 10  copies of DENV per mosquito: Chisq=9.13, df=4, P=0.0025) and high larval survival estimates ( FIG. 10  Mixed effects regression controlling for hatch order. Log 10  copies of DENV per mosquito: Chisq=8.38, df=4, P=0.0038), indicating that  Wolbachia -mosquito combinations that were better at blocking DENV were also inherently more fit. The strongest determinant of this difference appears to be the rate of egg laying. 
     It will be recognized from the foregoing that  Wolbachia  is a promising biological control agent against viruses including dengue, Zika and chikungunya within populations of the mosquito  Aedes aegypti.  Our aim was to understand the potential for the  Wolbachia -mediated pathogen blocking phenotype to persist over evolutionary time. We used artificial selection as a tool to tease apart genetic variation for this trait in  A. aegypti  and investigate its relationship with mosquito fitness. 
     The response to selection was rapid and resulted in populations that differed in blocking strength by 40%. This demonstrates that even within a single mosquito population carrying a recently introduced  Wolbachia  infection, there remains substantial genetic variation for blocking. The implications for field release are two-fold. First, blocking may exhibit phenotypic variation when the  Wolbachia  strains are crossed into local mosquito populations around the globe in preparation for local field releases. Second, the presence of variation means that blocking may evolve in the  Wolbachia  and/or  A. aegypti  populations through time post release. 
     Little is known about the selection pressures that may shape the evolutionary trajectory of  Wolbachia -mediated DENV blocking in  A. aegypti   27 . Empirical evidence shows that high  Wolbachia  density and thus blocking strength tend to associate with large fitness costs 19  and so it has been predicted that selection could favour reduced blocking over time 24 . Here, we found that  Wolbachia  density did correlate with blocking strength, however  Wolbachia -infected populations with stronger blocking had a higher intrinsic growth rate. For the first time, these data indicate the potential for stronger blockers to outcompete weaker blockers. Consistent with this result, populations selected for high blocking strength were most similar in phenotype and genotype to the Random and ancestral populations that were not subject to artificial selection. This suggests that high blocking genotypes are maintained at a high frequency in populations by natural selection. Published data on blocking stability 1 year after release trials in Australia show that blocking strength in field-collected mosquitoes was maintained at levels similar to the original lines 38 . Thus, our results suggest that this outcome is likely due to the maintenance of blocking by natural selection rather than a lack of genetic variation. 
     In the present disclosure, blocking strength was strongly associated with SNPs in the  A. aegypti  genome, demonstrating the capacity for the species to shape the nature of blocking. Crucially, the removal of  Wolbachia  from the evolved populations abolished the differences in blocking strength between the selection treatments, indicating that the genetic changes in the mosquito genome are only relevant in the context of an interaction with  Wolbachia.  This finding is in contrast with a recent study in  Drosophila melanogaster  natively infected with  Wolbachia  that found that evolutionary changes in host resistance explained most of the host adaptation to  Drosophila  C virus 45 . When we examined the identity of the genes within the  A. aegypti  genome that contained SNPs important for blocking strength, we found that they were not members of classical innate immune pathways (Toll, Imd, RNAi or JAK-STAT) 46 . Instead, they include a gene that encodes the glycoprotein cadherin that is important for cell-to-cell adhesion 39  and an alpha-mannisodase 2a enzyme which is involved in the N-glycan biosynthesis pathway.  Wolbachia  has been previously shown to alter the expression of genes involved in cell-cell adhesion and the N-glycan biosynthesis pathway 47 . The N-glycan biosynthesis pathway may be important as it is involved for the functioning of cadherin 40,41  and cadherin could be mediated in  Wolbachia&#39;s  interaction with the host cytoskeleton. Interestingly, DENV has been shown to bind cadherin within the cell 48  and could be a point of interaction between  Wolbachia,  DENV and  A. aegypti.  It is possible that  Wolbachia  is affecting the success of DENV by altering key molecules the virus needs for binding and entry into cells. Recent experimental work has suggested that the main impact of  Wolbachia  is at the point of limiting viral replication 49  however this work was carried out in cell culture where expression of genes involved in cell-to-cell adhesion could be altered. 
     Here, we used artificial selection as a tool to dissect genetic variation important for  Wolbachia -mediated DENV blocking in  A. aegypti.  These findings highlight the capacity for both  Wolbachia  and  A. aegypti  genomic variation to affect blocking strength. Promisingly, however, strong blocking was also associated with a faster mosquito population growth rate, which may help to drive and maintain the strength of  Wolbachia  mediated viral blocking over the long-term. At a mechanistic level, we have highlighted changes in the  A. aegypti  genome that most likely modify the strength of blocking and from a series of changes in the  Wolbachia  genome, developed a possible model to explain the symbiont&#39;s mode of action. Understanding mechanism may help evaluate and improve the specificity of  Wolbachia  strains against diverse mosquito genetic backgrounds. 
     SUPPLEMENTARY INFORMATION 
     Ethics Statement 
     All experiments in this study that utilised a human volunteer for mosquito blood-feeding were carried out at Monash University, Melbourne (Australia). The Monash University Human Research Ethics Committee gave ethical approval for the use of human volunteers to provide blood-meals to mosquitoes that were not infected with DENV (permit CF11/0766-2011000387). One volunteer was used throughout this study and provided written consent prior to the study. 
     Mosquitoes 
     We used a population of  Aedes aegypti  mosquitoes that were infected with the wMel (wMel.F) line of  Wolbachia  bacteria 21,53  and had since been maintained in the lab for 33 generations. Every 3 generations these mosquitoes were outcrossed with  Wolbachia -free mosquitoes collected from Queensland, Australia to maintain standing genetic variation that represent a natural population 24,53 . During outcrossing, females from the lab population were only allowed to mate with males from the natural populations to ensure the maternal transmission of  Wolbachia.  This is because  Wolbachia  bacteria are passed through the maternal line. 
     Dengue Virus 
     An isolate of DENV serotype 3 from Cairns was used in this study 54,55 . Virus was grown within C6/36  Aedes albopictus  cells following standard methods 24 . C6/36 cells were grown at 26° C. in T175 tissue culture flasks containing 25 ml RPMI 1640 media (Life Technologies, Carlsbad, Calif.) supplemented with 10% Fetal Bovine Serum (FBS, Life Technologies), 2% HEPES (Sigma-Aldrich, St. Louis, Mo.) and 1% Glutamax (Life Technologies). Prior to infection, C6/36 cells were grown to 80% confluency. At this point, the media was replaced with 25 ml RPMI supplemented with 2% FBS (Life Technologies), 2% HEPES (Sigma-Aldrich, St. Louis, Mo.) and 1% Glutamax (Life Technologies). 20 μl of a solution containing DENV-3 was added. After 7 days, the cells were scraped off and suspended in the media. The media was collected and centrifuged at 3200 g for 15 minutes at 4° C. The supernatant was then taken and frozen in single-use aliquots at −80° C. and all experiments in this paper were conducted using these aliquots. Viral titre was measured from a thawed aliquot by: 1) mixing 20 ul of the aliquot with 200 ul of TRIzol reagent (Invitrogen); 2) extracting the RNA following the manufacturer&#39;s protocol and treating with DNAse; and 3) quantifying DENV RNA using RT-qPCR (see section “Dengue virus quantification”). This was repeated 3 independent times for the same aliquot and an average viral titre was calculated. 
     Dengue Virus Quantification 
     The quantification of DENV was carried out via RT-qPCR using the LightCycler 408 (Roche). We used the TaqMan Fast Virus 1-Step Master Mix (ThermoFisher) in a total reaction volume of 10 ul, following the manufacturer&#39;s instructions 24 . The primers and probes used for DENV detection are listed in Table 3. The protocol for the RT-qPCR was as previously documented 56 . Data were analysed using absolute quantification where DENV copy number per sample was calculated from a reference curve. This reference curve was made up of known quantities of the genomic region of DENV that the primers amplify. This genomic region had previously been cloned into the pGEM-T plasmid (Promega, Madison, Wis.) and transformed into  Escherichia coli   56 . After growing this transformed  E. coli  in liquid LB overnight at 37° C. we then extracted the plasmid using the PureYield Plasmid Midiprep System kit (Promega) and linearized the plasmid by restriction digest. We then purified the plasmid using phenol-chloroform extraction, resuspended in 20 ul of UltraPure distilled water (Invitrogen) and quantified the plasmid by Qubit. A dilution series of 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2  and 10 1  copies of the viral genomic fragment were created and frozen as single-use aliquots. All assays measuring DENV load in this study used these identical aliquots and 3 replicates of the dilution series was run on every 96-well plate to create a reference curve for DENV quantification. For a given analysis, replicates from each population being compared were equally represented on each 96-well plate. 
       Wolbachia  Quantification 
     We measured the density of  Wolbachia  as the number of genome copies relative to the number of mosquito genome copies via multiplex qPCR on the LightCycler 408 (Roche) 57 . We used the LightCycler480 Probes Master mix (2× concentration from Roche) in a total reaction volume of 10 ul. The list of primers and probes are given in Table 3. The protocol for the RT-qPCR was as previously documented 56 . Basic relative quantification was used with mosquito genome copies as the reference and  Wolbachia  genome copies as the target. For a given analysis, replicates from each population being compared were equally represented on each 96-well plate. 
     Selection Experiment 
     We performed a bi-directional artificial selection experiment where we selected for increased and decreased DENV load (Low and High blocking treatments, respectively). We also included a random control treatment that imposed no directional selection (Random blocking treatment, see  FIG. 1 a   ). Each treatment was replicated with 3 independent lines 58  generated randomly from the same ancestral population of mosquitoes. 
     Each generation, mosquito eggs were hatched in trays (30×40×8 cm). Each tray contained 2 L of autoclaved RO water and 150-200 larvae. Larvae were fed with common fish food each day (Tetramin®, Melle, Germany). Rearing was performed under controlled conditions of temperature (26±2° C.), relative humidity (˜70%) and photoperiod (12:12, light:dark). After pupation, pupae were placed within 30×30×30 cm cages in cups containing autoclaved RO water for eclosion. At this stage, cages housed ˜450 individuals each. Dental wicks soaked in 10% sucrose water were placed in each cage as a food source. When mosquitoes were 5-7 days old the females from each line were allowed to blood-feed on the arm of a human volunteer in a random order. The next day, blood-engorged females were placed into separate cups enclosed with mesh (see  FIG. 1 b   ). Each cup contained filter paper sitting in autoclaved RO water to provide an oviposition site for female mosquitoes. A small petri dish was placed over the water to reduce the risk of mosquitoes drowning. A cotton wool ball soaked in 10% sucrose water was placed on top of the mesh as a food source for each cup. 
     After 4 days, the filter paper was collected from each female and dried following standard protocol for short-term egg storage 59 . Each set of eggs were numbered according to which mosquito they came from. On the same day, between 40 and 70 females from each population of the High and Low blocking lines were anaesthetised with CO 2  and injected with 3,903 genomic copies of dengue in 69 nl of RPMI media (5.66E−05 genomic copies/ml), delivered at a speed of 46 nl/sec into the thorax using a pulled glass capillary needle and a manual microinjector (Nanoject II, Drummond Sci.). The mosquitoes were then returned to individually labelled cups. Egg collection was done prior to injection to prevent the vertical transmission of DENV 60 . 
     At 7 days post infection, females were anaesthetised with CO 2  and were placed into individual wells in a 96-well plate containing 50 μl of extraction buffer. These samples were then homogenised with a 3 mm glass bead. Extraction buffer was made up of squash buffer (10 mM Tris pH 8.2, 1 mM EDTA, 50 mM NaCl) 61  with proteinase k at a concentration of 12.5 μl/ml (Bioline). Samples were then incubated for 5 minutes at 56° C. and 5 minutes at 95° C. We then examined DENV load per mosquito using RT-qPCR (see “Dengue virus quantification”). This method was used for rapid phenotype determination of a large number of samples. 
     Mosquitoes where then ranked in order from: the lowest DENV load in the High blocking lines; the highest DENV load in the Low blocking lines; and using a random number generator in the random line. Eggs from the top 20 mosquitoes were placed into separate cups of autoclaved RO water. The next day, larvae were then taken from cups in rank order until ˜200 larvae were collected for each replicate population. This was done to impose the strongest selection pressure as possible whilst ensuring enough mosquitoes will be reared for selection to also be possible in the subsequent generation. At this point, the passage protocol was repeated. In total, 4 rounds of selection were completed. 
     Dengue Virus Load and  Wolbachia  Density at Generation 4 
     After 4 rounds of selection, mosquitoes from each line were reared and injected with DENV as above (see “Selection protocol”). Seven days after injection, 30 mosquitoes from each line were dissected to separate the ovaries and the bodies since ovaries contain large densities of  Wolbachia  and could potentially mask patterns with DENV load in the body (ref?). Dissections were performed in 1× phosphate buffered saline (PBS) on a glass slide under a microscope using dissecting needles. Dissecting needles were soaked in 80% ethanol between each dissection and needles were changed between each line. Each body was placed into 1.5 ml tubes containing 200 ul of TRIzol reagent. Ovaries from 20 mosquitoes per line were collected in the same way. Each sample was then homogenised with a 3 mm glass bead and stored at −80° C. until used. 
     RNA was extracted from the TRIzol reagent for each mosquito body following the manufacturer&#39;s protocols and resuspended in 25 ul of UltraPure distilled water. Each sample was then treated with DNAse 1 (Sigma Aldrich) by adding lul of enzyme and 2.9 ul of buffer. 
     Samples were incubated at 37° C. for 30 minutes and then 75° C. for 10 minutes. At this point DENV quantification was carried out by RT-qPCR (see “Dengue load quantification”). DNA was also extracted from the TRIzol reagent for each mosquito body and set of ovaries collected following the manufacturer&#39;s protocols and resuspended in 25 ul of UltraPure distilled water. The density of  Wolbachia  was then measured using qPCR (see “ Wolbachia  quantification”). 
     Dengue Virus Load Over Time 
     At generations 0, 2 and 4 of the selection experiment, additional mosquitoes from the High and Low selection treatments and mosquitoes from the Random treatment were injected with DENV as above (see “Selection protocol”) to assess the change in DENV load over time. Seven days after injection, 10 mosquitoes from each line were collected in 1.5 ml tubes containing 200 ul of TRIzol reagent and homogenised with a 3 mm glass bead per sample. Samples were stored at −80° C. prior to RNA extraction. RNA extraction and DNAse treatment was carried out as above (see “Dengue virus load and  Wolbachia  density at generation 4”) and DENV load was quantified by RT-qPCR (see “Dengue load quantification”). 
     Role of  Wolbachia  in Phenotypic Differences 
     To confirm that the divergence in DENV load between treatments was as a result of  Wolbachia -mediated DENV blocking, we treated subpopulations of each line with the antibiotic tetracycline for 2 generations. Each generation, 10% sucrose water containing tetracycline (1.25 mg/ml tetracycline at pH 7 with unbuffered Tris) was given to adult mosquitoes 36  via dental wicks and replaced every 2 days. Control subpopulations of each line were kept separately and fed 10% sucrose at the adult stage. We then reared the lines for another generation with no antibiotic treatment to allow microbiota recovery. This is important since the microbiome can have important roles in mosquito resistance to arboviruses and we only want to measure the effect of  Wolbachia -mediated protection 57 . We transferred 100 ml of the larval rearing water from each control line to the corresponding antibiotic-treated line to re-introduce the resident microbiota, as is standard procedure 37 . This water was checked for egg and/or larval contamination. The following generation of mosquitoes (now 4 generations since the selection experiment) were then reared and injected with DENV as above (see “Selection experiment”) and collected in 1.5 ml tubes containing 200 ul of TRIzol reagent after 7 days of infection. These samples were homogenised with a 3 mm glass bead each and stored at −80° C. RNA and DNA extraction was carried out as above (“Dengue virus load and  Wolbachia  density at generation 4”); DENV load was quantified by RT-qPCR (see “Dengue virus quantification”); and  Wolbachia  density was quantified by qPCR (see  Wolbachia  quantification). 
     Genomic Analysis 
     DNA was extracted from 90 individual mosquitoes from each line at generation 4. We extracted DNA using the TRIzol reagent (Invitrogen), using a modified version of the manufacturer&#39;s protocol with additional washing steps using phenol, chloroform and isoamylalcohol (please see corresponding step-by-step methods on Nature&#39;s Protocol 
     Exchange). The DNA of 90 mosquitoes were pooled in equal volumes per line and sequenced using Illumina HiSeq3000 with 150 bp paired-end reads. 
     FastQC version 0.11.4 was used with default settings to check the quality of the raw reads. To minimise false positives, Trimmomatic version 0.36 was used to trim the 3′ ends if quality was &lt;20 and reads were discarded if trimming resulted in reads that were &lt;50 bp in length. We mapped the resulting reads to the  Wolbachia  genome AE017196.1 and the  Aedes agypti  assembly Liverpool AGWG-AaegL5 using BWA ALN and checked for quality using qualimap version 2.2.1. Indel realignment was completed using GATK version 3.8.0. Duplicates were removed using picard version 2.17.8 and poor quality maps were removed using samtools 1.6 and filtering via hex flags. The quality was checked using qualimap. SNPs were called using popoolation2. SNPs were then filtered with a minimum coverage of 20 and a maximum of 200. 
     Mosquito Life History Traits and Fitness Estimation 
     To understand the impact of the strength of  Wolbachia -mediated DENV blocking on mosquito fitness, we estimated population growth rates by calculating the per capita intrinsic rate of natural increase (r) for mosquitoes based on life history data collected from 3 replicate populations from each line of the High blocking, Low blocking and the Random blocking treatments. These included the median time to pupation, adult sex ratio, female adult daily survival over 3 bloodmeals and the size and timing of egg clutches over 3 bloodmeals. We used these data to construct Leslie matrix models to then calculate the asymptotic growth rate for a population that behaved exactly as the individuals observed in our experimental cages (models constructed as previously detailed 44 ). This approach assumes density independence and has been used as a holistic estimate to capture mosquito fitness in previous studies on mosquito fitness 44,62,63 . 
     We hatched offspring from mosquitoes at the end of the selection experiment by submerging eggs into autoclaved RO water and placing them into a vacuum chamber for 40 min. We used this reduced oxygen environment to induce synchronous hatching within each line to reduce variation in our data. We hatched lines in 3 batches, with each treatment being equally represented in each batch so that hatch time could be controlled for statistically. Hatched larvae were then separated from unhatched eggs and kept in trays of ˜200 larvae in 2 L of autoclaved RO water and were fed daily with Tetramin tablets. The number of larvae that had pupated each day was recorded per tray and pupae were placed in cups of water within separate 30×30×30 cm cages for each replicate. 
     Once all pupae emerged to the adult stage we measured sex ratio. We then transferred 60-80 females and 40 males per replicate into 20×20×30 cm cages to allow for mating. We gave each population the chance to take a blood meal from a human volunteer for 15 minutes in a randomised order. The next day, we placed cups containing filter paper and autoclaved RO water into each cage to allow for female oviposition. We changed these cups after 5 and 8 days and counted the number of eggs laid per cage within each time interval to get a measure of egg laying rate. We repeated this process 2 more times, resulting in 3 bloodmeals. After each bloodmeal we removed females that did not feed so that we had an accurate estimate of eggs laid per female. At the same time, we also measured female mortality, removing dead mosquitoes each day and censoring mosquitoes that we removed because they did not feed 64 . Accidental deaths and escapees were also recorded and censored from the dataset. 
     Eggs were counted using an adapted version of a previously determined protocol 65 . This protocol uses a high-resolution colour scanner to take images of the egg papers and creates a reference curve of manually counted eggs and the total area of an image that is black using ImageJ (see  FIG. 9 ). We used an Epson V39 flatbed scanner with 4800×4800 dpi. To ensure the highest accuracy, we carefully used a wet paintbrush to spread out eggs on the filter paper and we scanned each filter paper as they were still moist such that the eggs did not desiccate and change shape. Prior to using the ANALYZE: ANALYZE PARTICLES function in ImageJ, we set the upper threshold for calculating the total particle area at 95 by using the IMAGE:ADJUST:THRESHOLD function. 
     Statistical Analysis 
     All statistical analyses were performed in R version 3.2.2 (www.r-project.org/) and are listed in Table 2, along with sample sizes. Where multiple comparisons were made on a single data-set, P-values were corrected using the false discovery rate (FDR) method. For all mixed-effects models, the significance of fixed effects and their interactions was performed by sequentially removing model terms. Models were fit by maximum likelihood and statistically compared using a likelihood ratio test. We analysed log 10  copies of DENV per mosquito at generation 4 using mixed-effects models that included Treatment as a fixed effect and line, batch of RNA extraction and RT-qPCR plate as random factors ( FIG. 2 a   ). We analysed log 10  copies of DENV per mosquito over time using mixed-effects models that included Treatment, Generation and their interaction as fixed effects and line, batch of RNA extraction and RT-qPCR plate as random factors ( FIG. 2 b   ). We measured the effect of  Wolbachia  presence on log 10  copies of DENV per mosquito by using a mixed effects model in the presence of  Wolbachia  and another in the absence of  Wolbachia  ( FIG. 2 c   ). Both models included Treatment as the fixed effect and line, batch of RNA extraction and RT-qPCR plate as random factors. 
     We tested for the presence of a significant correlation between log 10  copies of DENV per mosquito and  Wolbachia  density in the bodies and the ovaries using a linear regression model with  Wolbachia  density as the independent variable ( FIG. 3  and  FIG. 8 , respectively). We tested for a correlation between log 10  copies of DENV per mosquito and the intrinsic growth rate of each line (with low and high juvenile survival separately) using mixed effects regression models including growth rate as the response variable, DENV load as the fixed effect and the order of hatching batch as a random effect ( FIG. 6  and data  FIG. 10 ). For each correlation, points represented population averages for each independent line. We calculated the reference curve for the automated counting of eggs using a linear regression model with the total particle area as the dependent variable and the number of eggs as the independent variable ( FIG. 9 ). 
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S., O&#39;Brochta, D. A., Carey, B. &amp; Atkinson, P. W. Assessing fitness costs for transgenic  Aedes aegypti  expressing the GFP marker and transposase genes.  Proc Natl Acad Sci USA  101, 891-896, doi:10.1073/pnas.0305511101 (2004).   64 Joy, T. K., Arik, A. J., Corby-Harris, V., Johnson, A. A. &amp; Riehle, M. A. The impact of larval and adult dietary restriction on lifespan, reproduction and growth in the mosquito  Aedes aegypti. Exp Gerontol  45, 685-690, doi:10.1016/j.exger.2010.04.009 (2010).   65 Mains, J. W., Mercer, D. R. &amp; Dobson, S. L. Digital image analysis to estimate numbers of  Aedes  eggs oviposited in containers.  J Am Mosq Control Assoc  24, 496-501, doi:10.2987/5740.1 (2008).   

     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Wolbachia genes containing SNPs differentiated between Low and High  
               
               
                 blocking. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 # 
                 min 
                 max 
                   
                   
                   
                   
               
               
                 SNPs 
                 P-value 
                 P-value 
                 Gene ID 
                 Gene 
                 Function 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 3 
                 4.55E−61 
                 5.32E−10 
                 WD_0095 
                   
                 D-alanine-D-alanine ligase 
                 yellow 
               
               
                   
                   
                   
                   
                   
                 Peptidoglycan biosynthesis 
                   
               
               
                 53 
                 1.77E−54 
                 0.00669482 
                 WD_Wp23SB 
                 23SB 
                 23S ribosomal subunit 
                 blue 
               
               
                   
                   
                   
                   
                   
                 Translation 
                   
               
               
                 1 
                 9.35E−43 
                 N.A. 
                 WD_1128 
                 murF 
                 UDP-N-acetylmuramoylalanyl- 
                 yellow 
               
               
                   
                   
                   
                   
                   
                 D-glutamyl-2,6- 
                   
               
               
                   
                   
                   
                   
                   
                 diaminopimelate-D-alanyl-D- 
                   
               
               
                   
                   
                   
                   
                   
                 alanyl ligase 
                   
               
               
                   
                   
                   
                   
                   
                 Peptidoglycan biosynthesis 
                   
               
               
                 7 
                 8.16E−37 
                 2.38E−25 
                 WD_tRNA- 
                 tRNA-Ile-1 
                 tRNA 
                 blue 
               
               
                   
                   
                   
                 Ile-1 
                   
                 Translation 
                   
               
               
                 34 
                 6.50E−21 
                 0.03017372 
                 WD_Wp16SA 
                 16SA 
                 16S ribosomal subunit 
                 blue 
               
               
                   
                   
                   
                   
                   
                 Translation 
                   
               
               
                 2 
                 1.12E−18 
                 2.43E−06 
                 WD_0928 
                 dnaK 
                 Chaperone protein, 
                 grey 
               
               
                   
                   
                   
                   
                   
                 Stress response 
                   
               
               
                 2 
                 1.03E−16 
                 1.31E−16 
                 WD_0112 
                 gyrB 
                 DNA gyrase, B subunit 
                 green 
               
               
                   
                   
                   
                   
                   
                 DNA topological change 
                   
               
               
                 4 
                 3.18E−14 
                 2.21E−07 
                 WD_0227 
                   
                 Elongation factor Tu family 
                 blue 
               
               
                   
                   
                   
                   
                   
                 protein 
                   
               
               
                   
                   
                   
                   
                   
                 Translation 
                   
               
               
                 4 
                 5.45E−12 
                 3.12E−05 
                 WD_0527 
                 uppS 
                 Undecaprenyl diphosphate 
                 yellow 
               
               
                   
                   
                   
                   
                   
                 synthase 
                   
               
               
                   
                   
                   
                   
                   
                 Peptidoglycan biosynthesis 
                   
               
               
                 1 
                 1.77E−10 
                 N.A. 
                 WD_tRNA- 
                 tRNA-Thr- 
                 tRNA 
                 blue 
               
               
                   
                   
                   
                 Thr-1 
                 1 
                 Translation 
                   
               
               
                 1 
                 5.49E−08 
                 N.A. 
                 WD_0154 
                 uvrC 
                 Excinuclease ABC, C subunit 
                 grey 
               
               
                   
                   
                   
                   
                   
                 DNA repair in response to 
                   
               
               
                   
                   
                   
                   
                   
                 damage 
                   
               
               
                 1 
                 1.62E−07 
                 N.A. 
                 WD_0613 
                   
                 Glycosyl transferase, group 1 
                 yellow 
               
               
                   
                   
                   
                   
                   
                 family protein 
                   
               
               
                   
                   
                   
                   
                   
                 Peptidoglycan biosynthesis 
                   
               
               
                 2 
                 1.83E−07 
                 0.04057723 
                 WD_1202 
                 gyrA 
                 DNA gyrase, A subunit 
                 green 
               
               
                   
                   
                   
                   
                   
                 DNA topological change 
                   
               
               
                 3 
                 4.43E−06 
                 6.28E−05 
                 WD_0317 
                 lon 
                 ATP-dependent protease La 
                 grey 
               
               
                   
                   
                   
                   
                   
                 Degradation of misfolded 
                   
               
               
                   
                   
                   
                   
                   
                 proteins 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Statistical analyses and sample sizes 
               
            
           
           
               
               
               
            
               
                 Figure 
                 Statistical test 
                 Sample size 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 2a 
                 DENV load by treatment (G4) 
                   
                 # Mosquitoes 
               
               
                   
                 Mixed effects model  
                 H1 
                 30 
               
               
                   
                 Random effects: Line; RNA extraction batch; and  
                 H2  
                 2 
               
               
                   
                 RT-qPCR plate  
                 H3  
                 30 
               
               
                   
                 Treatment: Chisq = 9.68, df = 7, P = 0.0079**  
                 L1  
                 13 
               
               
                   
                 Post-hoc Tukey comparisons:  
                 L2  
                 30 
               
               
                   
                 L-H: 0.0021**  
                 L3  
                 30 
               
               
                   
                 L-R: 0.0006***  
                 R1  
                 29 
               
               
                   
                 R-H: 0.94  
                 R2  
                 30 
               
               
                   
                   
                 R3  
                 30 
               
               
                 2b 
                 DENV load by treatment over time (G0, G2, G4) 
                   
                 # Mosquitoes 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Mixed effects model  
                 Generation: 
                 0 
                 2 
                 4 
               
               
                   
                 Random effects: Line; RNA extraction batch; and  
                 H1 
                 10 
                 10 
                 10 
               
               
                   
                 RT-qPCR plate  
                 H2  
                 10 
                 10 
                 10 
               
               
                   
                 Treatment: Chisq = 6.73, df = 7, P = 0.03*  
                 H3  
                 10 
                 10 
                 10 
               
               
                   
                 Generation: Chisq = 0.1, df = 9, P = 0.95  
                 L1  
                 10 
                 10 
                 2 
               
               
                   
                 Treatment * Generation: Chisq = 20.2, df = 13,  
                 L2  
                 10 
                 10 
                 10 
               
               
                   
                 P = 0.00046***  
                 L3  
                 10 
                 10 
                 10 
               
               
                   
                 Post-hoc Tukey comparisons: 
                 R1  
                 10 
                 10 
                 10 
               
               
                   
                   
                 R2  
                 10 
                 10 
                 10 
               
               
                   
                   
                 R3  
                 10 
                 10 
                 9 
               
            
           
           
               
               
               
               
            
               
                 2c 
                 DENY load with &amp; without  Wolbachia  (G8) 
                   
                 # Mosquitoes 
               
            
           
           
               
               
               
               
               
            
               
                   
                   Wolb − 
                 Treatment: 
                   Wolb − 
                   Wolb + 
               
               
                   
                 Mixed effects model  
                 H1 
                 9 
                 9 
               
               
                   
                 Random effects: Line; RNA extraction batch; and  
                 H2  
                 9 
                 9 
               
               
                   
                 qPCR plate  
                 H3  
                 5 
                 8 
               
               
                   
                 Treatment: Chisq = 1.25, df = 7, P = 0.53,  
                 L1  
                 9 
                 9 
               
               
                   
                 FDR-corrected P-value = 0.69  
                 L2  
                 8 
                 9 
               
               
                   
                 Values were removed from the  Wolb − treatment if  
                 L3  
                 9 
                 8 
               
               
                   
                   Wolbachia  showed (i.e. incomplete tetracycline curing).  
                 R1  
                 4 
                 9 
               
               
                   
                   Wolb +  
                 R2  
                 9 
                 9 
               
               
                   
                 Mixed effects model  
                 R3  
                 8 
                 7 
               
            
           
           
               
               
               
               
            
               
                   
                 Random effects: Line; RNA extraction batch; and  
                   
                   
               
               
                   
                 qPCR plate 
                   
                   
               
               
                   
                 Treatment: Chisq = 12.9, df = 7, P = 0.0016**  
                   
                   
               
               
                   
                 FDR-corrected P-value = 0.0032**  
                   
                   
               
               
                   
                 Post-hoc Tukey comparisons:  
                   
                   
               
               
                   
                 H-R: P = 0.95  
                   
                   
               
               
                   
                 R-L: P &lt; 1e−05***  
                   
                   
               
               
                   
                 H-L: P = 2.14e−05 *** 
                   
                   
               
               
                 3 
                 Correlation of DENY load and  Wolbachia  density in  A.   
                   
                 # Mosquitoes 
               
               
                   
                   aegypti  bodies (G4)  
                 H1 
                 29 
               
               
                   
                 Linear regression model  
                 H2  
                 29 
               
               
                   
                   Wolbachia  density: t = −2.9, df = 7, P = 0.02*, R 2  = 0.55  
                 H3  
                 30 
               
               
                   
                 Log 10  copies of DENV are averages of data presented in  
                 L1  
                 13 
               
               
                   
                 FIG. 2a.  Wolbachia  density was measured from the same  
                 L2  
                 30 
               
               
                   
                 mosquitoes.  
                 L3  
                 29 
               
               
                   
                   
                 R1  
                 29 
               
               
                   
                   
                 R2  
                 30 
               
               
                   
                   
                 R3  
                 29 
               
               
                 6 
                 Correlation of  Aedes aegypti  population growth rate (G5)  
                   
                 # Larvae  
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 with DENY load (G4) with low juvenile survival  
                 Tray: 
                 1 
                 2 
                 3 
               
               
                   
                 Population growth rate data was calculated using Leslie  
                 H1 
                 200 
                 206 
                 203 
               
               
                   
                 matrix models using data from 3 replicate populations  
                 H2  
                 194 
                 187 
                 190 
               
               
                   
                 per line per treatment (same as data FIG. 10) with  
                 H3  
                 201 
                 191 
                 199 
               
               
                   
                 juvenile survival estimated at 43%.  
                 L1  
                 192 
                 193 
                 189 
               
               
                   
                 Mixed effects regression model  
                 L2  
                 192 
                 191 
                 191 
               
               
                   
                 Random factors: hatch order  
                 L3  
                 211 
                 187 
                 222 
               
               
                   
                 Log 10  copies of DENV per mosquito: Chisq = 9.13,  
                 R1  
                 194 
                 208 
                 213 
               
               
                   
                 df = 4, P = 0.0025**  
                 R2  
                 201 
                 194 
                 189 
               
               
                   
                 FDR-corrected P-value: 0.0038**  
                 R3  
                 213 
                 214 
                 200 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 # Mosquitoes 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 Cage: 
                 1 
                 2 
                 3 
               
               
                   
                   
                 H1 
                 86 
                 79 
                 66 
               
               
                   
                   
                 H2  
                 82 
                 65 
                 73 
               
               
                   
                   
                 H3  
                 35 
                 80 
                 64 
               
               
                   
                   
                 L1  
                 90 
                 59 
                 39 
               
               
                   
                   
                 L2  
                 81 
                 80 
                 72 
               
               
                   
                   
                 L3  
                 79 
                 83 
                 73 
               
               
                   
                   
                 R1  
                 78 
                 78 
                 77 
               
               
                   
                   
                 R2  
                 81 
                 67 
                 78 
               
               
                   
                   
                 R3  
                 87 
                 82 
                 54 
               
            
           
           
               
               
               
            
               
                 Extd  
                   Wolbachia  densities of samples treated or untreated with  
                 Same as FIG. 2c 
               
            
           
           
               
               
               
               
            
               
                 data 1  
                 tetracycline (G8)  
                   
                   
               
               
                   
                   Wolbachia  density was measured from the same  
                   
                   
               
               
                   
                 mosquitoes as in FIG. 2c.  
                   
                   
               
               
                 Extd 
                 Correlation of DENY load and  Wolbachia  density in  A .  
                   
                 # Mosquitoes 
               
               
                 data 2 
                   aegypti  ovaries (G4)  
                 H1 
                 11 
               
               
                   
                 Linear regression model  
                 H2  
                 11 
               
               
                   
                   Wolbachia  density: t = −0.56, df = 7, P = 0.59,  
                 H3  
                 12 
               
               
                   
                 R 2  = 0.042 
                 L1  
                 6 
               
               
                   
                 Log 10  copies of DENV are averages of data presented in  
                 L2  
                 11 
               
               
                   
                 FIG. 2a.  Wolbachia  density was measured from the same  
                 L3  
                 12 
               
               
                   
                 mosquitoes. 
                 R1  
                 12 
               
               
                   
                   
                 R2  
                 12 
               
               
                   
                   
                 R3  
                 12 
               
            
           
           
               
               
               
            
               
                 Extd 
                 Reference curve for estimation of egg numbers 
                 14 egg papers of variable  
               
               
                 data 3 
                 Linear regression model  
                 density  
               
            
           
           
               
               
               
               
            
               
                   
                 Egg number: t value = 49.426, df = 2, P = 3.08e−15 ***,  
                   
                   
               
               
                   
                 R 2  = 0.995  
                   
                   
               
               
                   
                 Y = 183.1547X-8596.8768  
                   
                   
               
            
           
           
               
               
               
            
               
                 Extd 
                 Correlation of  Aedes aegypti  population growth rate (G5)  
                 Same as 4 
               
            
           
           
               
               
               
               
            
               
                 data 4 
                 with DENY load (G4) with high juvenile survival  
                   
                   
               
               
                   
                 Population growth rate data was calculated using Leslie  
                   
                   
               
               
                   
                 matrix models using data from 3 replicate populations  
                   
                   
               
               
                   
                 per line per treatment (same as FIG. 6) with juvenile  
                   
                   
               
               
                   
                 survival estimated at 92%.  
                   
                   
               
               
                   
                 Mixed effects regression model  
                   
                   
               
               
                   
                 Random factors: hatch order.  
                   
                   
               
               
                   
                 Log 10  copies of DENV per mosquito: Chisq = 8.38,  
                   
                   
               
               
                   
                 df = 4, P = 0.0038**  
                   
                   
               
               
                   
                 FDR-corrected P-value: 0.0038** 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Primers and probes 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Genomic 
                   
                 5′-3′ 
                   
               
               
                 Target 
                 region 
                 Direction 
                 sequence 
                 Tm 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Dengue 
                 3′UTR 
                 Fw 
                 AAGGACTAGAGG 
                 54 
               
               
                 virus 
                   
                   
                 TTAGAGGAGACCC 
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 11) 
                   
               
               
                   
                   
                 Rv 
                 CGTTCTGTGCCT 
                 58 
               
               
                   
                   
                   
                 GGAATGATG 
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 12) 
                   
               
               
                   
                   
                 Probe 
                 FAM-AACAGCATAT 
                   
               
               
                   
                   
                   
                 TGACGCTGGGAGAG 
                   
               
               
                   
                   
                   
                 ACCAGA-BHQ1/3 
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 18) 
                   
               
               
                   
               
               
                 
                   Aedes 
                 
                 AAEL004175 
                 Fw 
                 TCCGTGGTATCTC 
                 60 
               
               
                 
                   aegypti 
                 
                 (RPS17) 
                   
                 CATCAAGCT 
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 13) 
                   
               
               
                   
                   
                 Rv 
                 CACTTCCGGCACG 
                 60 
               
               
                   
                   
                   
                 TAGTTGTC 
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 14) 
                   
               
               
                   
                   
                 Probe 
                 FAM-CAGGAGGAG 
                   
               
               
                   
                   
                   
                 GAACGTGAGCGC 
                   
               
               
                   
                   
                   
                 AG-BHQ1/3 
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
               
               
                   
                   
                   
                 NO:19) 
                   
               
               
                   
               
               
                 
                   Wolbachia 
                 
                 WD0513 
                 Fw 
                 CAAATTGCTCTTG 
                 60 
               
               
                 
                   pipientis 
                 
                   
                   
                 TCCTGTGG 
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 15) 
                   
               
               
                   
                   
                 Rv 
                 GGGTGTTAAGCAG 
                 60 
               
               
                   
                   
                   
                 AGTTACGG 
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 16) 
                   
               
               
                   
                   
                 Probe 
                 LC640-TGAAATG 
                   
               
               
                   
                   
                   
                 GAAAAATTGGCGA 
                   
               
               
                   
                   
                   
                 GGTGTA 
                   
               
               
                   
                   
                   
                 GG-3′Iowablack 
                   
               
               
                   
                   
                   
                 RQ-Sp 
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 17) 
               
               
                   
               
            
           
         
       
     
     The disclosure has been illustrated by the previous examples. Variations and modification of the specific techniques and approaches described herein will be apparent to those skilled in the art, given the benefit of the present disclosure, and are included in the scope of this invention.