Transcriptomic Analysis of Aedes aegypti Innate Immune System in Response to Ingestion of Chikungunya Virus

Aedes aegypti (L.) is the primary vector of emergent mosquito-borne viruses, including chikungunya, dengue, yellow fever, and Zika viruses. To understand how these viruses interact with their mosquito vectors, an analysis of the innate immune system response was conducted. The innate immune system is a conserved evolutionary defense strategy and is the dominant immune system response found in invertebrates and vertebrates, as well as plants. RNA-sequencing analysis was performed to compare target transcriptomes of two Florida Ae. aegypti strains in response to chikungunya virus infection. We analyzed a strain collected from a field population in Key West, Florida, and a laboratory strain originating from Orlando. A total of 1835 transcripts were significantly expressed at different levels between the two Florida strains of Ae. aegypti. Gene Ontology analysis placed these genes into 12 categories of biological processes, including 856 transcripts (up/down regulated) with more than 1.8-fold (p-adj (p-adjust value) ≤ 0.01). Transcriptomic analysis and q-PCR data indicated that the members of the AaeCECH genes are important for chikungunya infection response in Ae. aegypti. These immune-related enzymes that the chikungunya virus infection induces may inform molecular-based strategies for interruption of arbovirus transmission by mosquitoes.


Introduction
Aedes aegypti (L.) is a major vector of arboviruses including chikungunya, dengue, yellow fever, and Zika. Chikungunya virus (CHIKV) is an emerging viral disease in the family Togaviridae, genus Alphavirus, which is transmitted to humans by infected mosquitoes, primarily Ae. aegypti and Ae. albopictus. Recent outbreaks of CHIKV occurred in Kenya in 2004 (East/Central/South African, ECSA, CHIKV lineage) and the island of La Réunion in 2005-2006 (Indian Ocean CHIKV lineage). CHIKV outbreaks were also documented in Gabon on the west coast of Central Africa (East Central South Africa lineage) in 2007 and in 2010 [1]. Later, CHIKV emerged in the New World in 2013 on St. Martin Island (Asian CHIKV lineage) and continued to spread throughout the Americas [2][3][4]. Other Aedes species that have been reported to transmit CHIKV include Ae. vittatus and Ae. koreicus. Aedes vittatus has a geographical distribution throughout tropical Asia, Africa, and the Mediterranean region of Europe [5], while Ae. koreicus is new to Europe with a geographical distribution consisting of tropical and sub-tropical climates across the globe [6]. More than four million cases of human infection involving CHIKV have occurred worldwide over the past 12 years, making mosquito control and interruption

Global Changes in Transcriptome of the Aedes aegypti Female Adult in Response to CHIKV Infection
Ae. aegypti is genetically polymorphic, as shown by differences among distinct geographic populations (strains). To understand the molecular interactions of the arbovirus with a natural population of Ae. aegypti from Florida, RNA-sequencing was conducted to explore the global changes in the Ae. aegypti (Key West and Orlando populations/strains) transcriptome in response to oral ingestion of CHIKV-infected blood and subsequent infection. In this study, four-day-old adult females of Ae. aegypti were fed a blood meal containing 8.0 ± 0.09 and 8.3 ± 0.08 log10 plaque forming units (pfu)/mL of CHIKV (Table 1). Fresh-fed (3 h post infection) mosquitoes ingested 4.89 to 5.68 log10 pfu/mL of CHIKV. The highest mosquito body titers observed for both strains occurred 5 days post infection (Key West, 5.87 ± 0.39 and Orlando, 4.78 ± 0.39 log10 pfu/mL, Table 1). A two-way ANOVA showed a significant strain (F 1,59 = 26.04, p < 0.0001), time (F 5,59 = 17.40, p < 0.0001), and strain by time interaction (F 5,59 = 5.85, p = 0.0002). Pairwise comparisons between time points for the Key West strain showed significantly higher mosquito body viral titers at 5 days and 7 days post infection compared to 1 day post infection ( Table 1). The remaining comparisons of the time points for the Key West strain were not significantly different from one another. Pairwise comparisons between time points for the Orlando strain had significantly higher mosquito body viral titers at 5 days post infection compared to 1 day post infection. Viral titers at 2 days post infection were lower than all other time points (Table 1). Comparisons between strains at each time point (e.g., Key West versus Orlando at 1 day post infection) showed similar titers for all time points except for 2 days post infection, where the Orlando strain had significantly lower viral titer than the Key West strain (Table 1). Table 1. Chikungunya virus (CHIKV) titers (log10 pfu/mL) (Least Squares means ± Standard Error) in infectious blood meals and mosquitoes of the Key West and Orlando strains of Aedes aegypti. Titers followed by the same letter are not significantly different from one another. Comparisons of time points are presented only for Key West (lower case letters) and Orlando (upper case letters) strains.

Expression Profiles of DE (Differentially Expressed) Transcripts in Response to Chikungunya Virus (CHIKV) Infection in Aedes aegypti Key West Population/Strain
RNA was extracted from female Ae. aegypti for the time course study, first at three hours post ingestion and again at three days post ingestion (dpi). A total of 18 RNA-seq libraries were created using female Ae. aegypti infected by CHIKV (3 h and 3 dpi) and the control (fed uninfected blood, 3 h and 3 dpi), using three replicates of each group. A total of 706,051,842 raw reads were generated from the Orlando strain. The cleanup resulted in 187,384,965 cleaned reads, which mapped to 18,840 transcripts of Ae. aegypti (Table S1). A total of 797,720,788 raw reads were generated from the Key West strain. The cleanup resulted in 797,360,677 cleaned reads, which mapped to 18,840 transcripts of Ae. aegypti (Table S1).
Functional analysis based on Gene Ontology was conducted on the significant differentially expressed (DE) transcripts in response to CHIKV ingestion in the Ae. aegypti Key West population/strain. Analysis of mRNA expression profiles of Ae. aegypti infected with CHIKV at different time points revealed a relatively high number of DE transcripts 3 h after blood-feeding. There were 2516 DE genes (p-adj (p-adjust value) ≤ 0.01), including 1299 upregulated and 1217 downregulated genes at 3 h post infection with CHIKV ( Figures 1A and S1A). An average of 35% of the transcripts were classified as an unknown function group (35.3% in the total: 32.0% in the Up, 38.7% in the Down). The remaining DE transcripts were matched to the functional categories of Binding (19.4% in the total: 19.0% in the Up, 20% in the Down), Catalytic activity (13.3% in the total: 15.7% in the Up, 10.7% in the Down), Cellular process (9.0% in the total: 10.3% in the Up, 7.5% in the Down), Immune system process (0.11% in the total: 0.4% in the Up, 0.2% in the Down), Metabolic process (12.6% in the total: 15.1% in the Up, 10.0% in the Down), Response to stimulus (2.2% in the total: 1.8% in the Up, 2.6% in the Down), Regulation of biological process (3.0% in the total: 2.4% in the Up, 3.7% in the Down), Structural molecular activity (0.7% in the total: 0.8% in the Up, 0.6% in the Down), Transporter activity (3.0% in the total: 2.6% in the Up, 3.3% in the Down), Developmental process (0.4% in the total: 0.3% in the Up, 0.4% in the Down), and Signal transducer activity (1.1% in the total: 0.3% in the Up, 2.0% in the Down).    Figures 1B and S1B). Most of these transcripts (30.7% in the total: 34.3% in the Up, 29.3% in the Down) had unknown functions. The remaining DE transcripts were mainly matched to the functional categories of Binding (21.7% in the total: 13.1% in the Up, 25.1% in the Down), Catalytic activity (10.9% in the total: 12.7% in the Up, 10.1% in the Down), Cellular process (13.7% in the total: 15.7% in the Up, 13.0% in the Down), Immune system process (0.07% in the total: 0.3% in the Up, 0.0% in the Down), Metabolic process (12.6% in the total: 19.2% in the Up, 9.9% in the Down), Response to stimulus (3.0% in the total: 0.5% in the Up, 4.0% in the Down), Regulation of biological process (4.5% in the total: 1.7% in the Up, 5.7% in the Down), Structural molecular activity (2.0% in the total: 6.2% in the Up, 0.3% in the Down), Transporter activity (1.5% in the total: 2.4% in the Up, 1.2% in the Down), Developmental process (0.4% in the total: 0.0% in the Up, 0.06% in the Down), and Signal transducer activity (0.7% in the total: 0.0% in the Up, 0.9% in the Down).  18.1% in the Down), Catalytic activity (9.9% in the total: 10.3% in the Up, 9.4% in the Down), Cellular process (10.6% in the total: 12.6% in the Up, 7.8% in the Down), Immune system process (0.0% in the total: 0.0% in the Up, 0.0% in the Down), Metabolic process (10.0% in the total: 10.0% in the Up, 10.0% in the Down), Response to stimulus (3.0% in the average: 4.1% in the Up, 1.4% in the Down), Regulation of biological process (4.1% in the total: 5.5% in the Up, 2.2% in the Down), Structural molecular activity (0.7% in the total: 0.8% in the Up, 0.6% in the Down), Transporter activity (1.7% in the total: 1.9% in the Up, 1.4% in the Down), Developmental process (0.5% in the total: 0.5% in the Up, 0. 6% in the Down), and Signal transducer activity (1.0% in the total: 1.5% in the Up, 0.2% in the Down).

Expression Profiles of the DE Transcripts in Response to CHIKV Infection in the Key West and Orlando Strains of Aedes aegypti
Comparison of the transcriptome profiles of the Key West and Orlando Ae. aegypti strains in response to CHIKV 3 dpi revealed 1,835 DE transcripts (p-adj ≤ 0.01, 860 upregulated and 975 downregulated, Figures 1D and S1D). Most of the DE transcripts (36.8% in the total: 33.5% in the Up, 39.8% in the Down) had unknown functions ( Figures 1D and S1D). The other matched functional categories included binding (18.5% in the total: 18.2% in the Up, 18.7% in the Down), catalytic activity (9.9% in the total: 9.9% in the Up, 9.9% in the Down), cellular process (11.7% in the total: 13.6% in the Up, 9.9% in the Down), immune system process (0.07% in the total: 0.05% in the Up, 0.09% in the Down), metabolic process (12.0% in the total: 14.4% in the Up, 9.9% in the Down), response to stimulus (2.4% in the total: 1.7% in the Up, 3.0% in the Down), regulation of biological process (3.8% in the total: 3.7% in the Up, 3.9% in the Down), structural molecular activity (1.5% in the total: 2.8% in the Up, 0.4% in the Down), transporter activity (2.3% in the total: 1.5% in the Up, 3.0% in the Down), developmental process (0.3% in the total: 0.1% in the Up, 0.5% in the Down), and signal transducer activity (0.7% in the total: 0.5% in the Up, 0.8% in the Down).

Expression Profiles of the DE Transcripts in Response to CHIKV Infection in Orlando Aedes aegypti Strain
Analysis of mRNA expression profiles of the Ae. aegypti mosquitoes infected with CHIKV from the Orlando strain detected a relatively high number of DE transcripts 3 days post injection. There were 675 DE genes (p-adj ≤ 0.01), including 336 upregulated and 339 downregulated genes (Figures 1E and S1E). Most of these transcripts (99.1% in the total: 18.2% in the Up, 29.3% in the Down) had unknown functions. Among these downregulated genes, only three genes were determined in GO analysis, which was not shown in Figure 1. The remaining DE transcripts mainly matched to the functional categories of binding (7.5% in the total: 15.1% in the Up, 0% in the Down), catalytic activity (7.6% in the total: 15.3% in the Up, 0% in the Down), cellular process (3.5% in the total: 7.0% in the Up, 0.0% in the Down), immune system process (0.2% in the total: 0.4% in the Up, 0.0% in the Down), metabolic process (7.7% in the total: 15.4% in the Up, 0.0% in the Down), response to stimulus (1.4% in the total: 2.9% in the Up, 0.0% in the Down), regulation of biological process (1.4% in the total: 2.9% in the Up, 0.0% in the Down), structural molecular activity (0.2% in the total: 0.4% in the Up, 0.0% in the Down), transporter activity (1.3% in the total: 2.6% in the Up, 0.0% in the Down), developmental process (0.2% in the total: 0.4% in the Up, 0.0% in the Down), and signal transducer activity (0.6% in the total: 1.1% in the Up, 0.0% in the Down).

Changes of the Immune-Related Genes in the Female Adult of the Key West Strain of Ae. aegypti in Response to CHIKV Infection
For the Key West population/strain of Ae. aegypti, 3 h post ingestion of CHIKV infected blood a total of 1112 transcripts showed 1.8 or more log2-fold changes (p-adj ≤ 0.01, upregulated 456, downregulated 656). Eighty-nine immune-related DE transcripts were significantly upregulated in response to CHIKV after 3 h, compared to the Key West control group. These up-or down-regulated immunity-related genes encoded two allergen, one caspase-1 protein, one cecropin anti-microbial peptide (AAEL000621-RA), five Class B Scavenger Receptor, seven Clip-Domain Serine Protease family B and E, six C-Type Lectin, two fibrinogen and fibronectin, one Gram-Negative Binding Protein, one lachesin, five leucine-rich immune protein, one Leucine-rich repeat-containing protein, two Leucine-rich repeat-containing protein, one SEC14 protein, one serine collagenase 1 precursor, one serine hydroxymethyl transferase, three serine protease, four serine protease inhibitor (serpin), twelve serine-type endopeptidase, four sidestep protein, one signal peptide peptidase, one tep2 protein and one tep3 protein (AAEL008607), one toll protein, two toll-like receptor, twenty-two trypsins, and one venom allergen (Table 2). Table 2. Immune-related genes that were significantly regulated in the Key West strain of Aedes aegypti 3 h post infection with CHIKV compared with Control in Key West strain (p-adj ≤ 0.01, log2-fold change ≥ ± 1.8).  When individuals of the Key West strain of Ae. aegypti were tested with CHIKV at 3 dpi, a total of 343 DE transcripts showed ≥ ±1.8 log2-fold changes (p-adj ≤ 0.01, upregulated 118, downregulated 225). Twenty-five immunity-related DE transcripts were significantly upregulated in response to CHIKV 3 dpi. These up-or down-regulated immunity-related genes encoded one cecropin anti-microbial peptide (AAEL004223), three Clip-Domain Serine Protease family B, three C-Type Lectins (CTL), one defensin anti-microbial peptide, one fibrinogen and fibronectin, one leucine-rich immune protein, three leucine-rich transmembrane protein, one posF21, putative protein, one SEC14 putative protein, one serine/threonine protein kinase, four serine-type endopeptidase, one Trypsin 3A1 Precursor, three trypsin, and one vav1 protein (Table 3). Table 3. Immune-related genes that were significantly regulated in the Key West strain of Aedes aegypti 3 days post ingestion with CHIKV compared with Control in Key West strain (p-adj ≤ 0.01, log2-fold change ≥ ± 1.8). When the Orlando strain Ae. aegypti were infected with CHIKV at 3 dpi, 316 DE transcripts had ≥±1.8 log2-fold changes (p-adj ≤ 0.01, 314 upregulated, 2 downregulated). Thirty-two immunity-related DE transcripts were significantly upregulated in response to CHIKV 3 dpi compared to the Orlando control group. These immunity-related genes encoded two cecropin anti-microbial peptide (AAEL004223 and AAEL017211), one chymotrypsin, two C-Type Lectins (CTL), one C-Type Lysozyme, one defensin anti-microbial peptide, four fibrinogens and fibronectins, one gambicin anti-microbial peptide, one leucine-rich repeat protein, one leucine-rich transmembrane protein, three Serine Protease Inhibitors (serpin), five serine-type endopeptidase, two Trypsin 3A1 Precursor, six trypsin, and two venom allergen (Table 4).

Changes of the Immunity-Related Genes of the Female Adults of Ae. aegypti between the Key West and Orlando Strains in Response to CHIKV Infection
When the Key West strain was compared with the Orlando strain among individuals following 3 dpi with CHIKV, a total of 856 transcripts had 1.8 log2-fold or above changes (p-adj ≤ 0.01, upregulated 258, downregulated 598). Thirty-three immunity-related DE transcripts were significantly up/down-regulated in response to CHIKV 3 dpi when Key West CHIKV-infected mosquitoes were compared to Orlando CHIKV-infected individuals. These up-or down-regulated immunity-related genes encoded two allergen, one br serine/threonine-protein kinase, one cecropin anti-microbial peptide (AAEL017211), three Class B Scavenger Receptors, one Clip-Domain Serine Protease family B, two C-Type Lectins (CTL), one D7 protein, three fibrinogen and fibronectin, one Gram-Negative Binding Protein, one lachesin, one leucine-rich repeat protein, two leucine-rich transmembrane protein, three Serine Protease Inhibitor (serpin), one serine protease, one serine-pyruvate aminotransferase, one serine-type endopeptidase, one TOLL pathway signaling, one toll protein, two Toll-like receptor, two trypsins, and two venom allergens (Table 5). When mosquitoes of the Key West strain were compared with the Orlando strain of Ae. aegypti 3 dpi, a total of 414 transcripts had 1.8 or above log2-fold changes (p-adj ≤ 0.01, upregulated 219, downregulated 195). Thirteen immunity-related DE transcripts in the Key West strain were significantly regulated in response to ingestion of the CHIKV-infected blood at 3 dpi compared with the Orlando strain. These up-or down-regulated immunity-related genes encoded one autophagy-related gene, one fibrinogen and fibronectin, one Gram-Negative Binding Protein, one JAKSTAT pathway signaling Signal, one protein serine/threonine kinase, one scavenger receptor, one serine protease, one serine/threonine protein kinase, two serine-type endopeptidases, two trypsins, and one viral IAP-associated factor ( Table 6). To characterize Ae. aegypti defensin and other immune-related genes in response to CHIKV exposure, we measured AaeDEFA, AaeDEFD, AaeDEFa, AaeDNR1, AaeCECH, and AaeTEP3 expressions in orally ingested Ae aegypti. Multivariate analysis of variance (MANOVA) showed the significant effects of strain of Ae. aegypti, time, and their interaction. For the significant strain effect, standardized canonical coefficients (SCCs) showed that AaeTEP3 was the primary contributor with approximately 2-16-fold greater effect than all other genes (Table 7). SCCs showed that AaeDEFA had the second highest contribution to the significant strain effect with approximately 1.3-8-fold greater effect than the other genes (Table 7). SCCs showed that AaeDEFa had the third highest contribution to the significant strain effect with about 3-6-fold greater effect than the other genes ( Table 7). Expression of AaeDEFa was negatively correlated with expression of AaeTEP3 and AaeDEFA. Differential expression of AaeTEP3, AaeDEFa, and AaeDEFa were all much higher for the Key West than Orlando strain of Ae. aegypti (Figure 2A-D).
For both the significant time and interaction effects, SCCs showed that AaeDEFA was the primary contributor (time, 2-11-fold, interaction, 2-44-fold) with AaeDEFD (time, 2-6-fold, interaction, 2-24-fold) and AaeTEP3 (time, 2-6-fold, interaction, 2-20-fold) having similar and the second highest contribution to the significant time and interaction effects. SCCs showed that AaeCECH was the third highest contributor (time, 2-4-fold, interaction, 2-13-fold) to the significant time and interaction effects. For the time effect, AaeTEP3 was negatively correlated with the expression of these three other genes. For the interaction, AaeCECH was negatively correlated with the expression of these three other genes (Table 7).
Because the interaction was significant, we focused on pairwise comparisons of strain by time combinations. Specifically, we compared the two strains of Ae. aegypti at each distinct time period (e.g., Key West versus Orlando at 3 h post infection), resulting in less than all possible comparisons. Gene expression of AaeDEFA was significantly different between the two strains of Ae. aegypti except at 24 and 240 h post infection. Gene expression of AaeDEFA was 2-9-fold significantly higher for the Key West than the Orlando strains of Ae. aegypti at most time points, except 120 and 168 h, where Orlando was 4-fold significantly higher. The general pattern of expression of AaeDEFA was characterized by an increase from 3 h post infection to the highest expression at 72 h for Key West and 120 h for Orlando, followed by a sharp decline in expression at later periods for both strains of Ae. aegypti (Figure 2A,B). Gene expression of AaeDEFD was 2-7-fold significantly higher for the Key West than the Orlando strain of Ae. aegypti at 3, 48, 72, and 240 h post infection. Gene expression of AaeDEFD was 3-15-fold significantly higher for the Orlando than the Key West strain of Ae. aegypti at 120 h and 168 h post infection. The two strains did not differ in expression of AaeDEFD at 24 h post infection. The highest gene expression of AaeDEFD was observed 72 h and 120 h post ingestion for the Key West and the Orlando strains, respectively. Gene expression of AaeDEFD declined after these periods for both Ae. aegypti strains (Figure 2A,B). Similarly, gene expression of AaeTEP3 was 4-22-fold significantly higher for the Key West than the Orlando strain of Ae. aegypti at all times, except 3 and 48 h post ingestion ( Figure 2C,D). Gene expression of AaeCECH was similar for both Ae. aegypti strains at most time points. However, AaeCECH was 2-11-fold significantly higher for the Key West than the Orlando strains of Ae. aegypti at 48, 72, and 240 h post ingestion ( Figure 2C,D). In summary, AaeDEFA, AaeDEFD, AaeDEFa, AaeDNR1, AaeCECH, and AaeTEP3 expressions were significantly higher for most times post ingestion for the Key West strain, except for 120 and 168 h post ingestion when expression of AaeDEFA and AaeDEF was higher for the Orlando strain of orally infected Ae. aegypti. highest gene expression of AaeDEFD was observed 72 hours and 120 hours post ingestion for the Key West and the Orlando strains, respectively. Gene expression of AaeDEFD declined after these periods for both Ae. aegypti strains (Figures 2a and 2b). Similarly, gene expression of AaeTEP3 was 4-22-fold significantly higher for the Key West than the Orlando strain of Ae. aegypti at all times, except 3 and 48 hours post ingestion (Figures 2c and 2d). Gene expression of AaeCECH was similar for both Ae. aegypti strains at most time points. However, AaeCECH was 2-11-fold significantly higher for the Key West than the Orlando strains of Ae. aegypti at 48, 72, and 240 hours post ingestion (Figures 2c and  2d). In summary, AaeDEFA, AaeDEFD, AaeDEFa, AaeDNR1, AaeCECH, and AaeTEP3 expressions were significantly higher for most times post ingestion for the Key West strain, except for 120 and 168 hours post ingestion when expression of AaeDEFA and AaeDEF was higher for the Orlando strain of orally infected Ae. aegypti.

Discussion
Understanding the Aedes vector-chikungunya virus interactions of natural and geographically distinct populations is fundamentally important since it may enable the search for new methods and strategies for interrupting arbovirus transmission. Previous transcriptomic studies have shown altered pathways in response to viral infection between genetically distinct strains of mosquito species [29,30]. In the current study, transcripts enriched in CHIKV infected females encoded proteins associated with functions which are largely unknown, binding catalytic activities, cellular process, metabolism process, response to stimulus, regulation of biological process, and immune system process (Figures 1  and S1). A previous study disclosed that serine-type endopeptidases and trypsins were significantly upregulated in mosquitoes following CHIKV ingestion [17]. Our transcriptomic analysis also showed that serine-type endopeptidases and trypsins were significantly upregulated in Ae. aegypti following CHIKV 3 days post infection in the Key West strain and Orlando strains. Comparison between the Key West and Orlando strains 3 days post infection with CHIKV showed that both serine-type endopeptidases and trypsins were significantly downregulated.
Numerous reports have shown altered expression of cecropin anti-microbial peptide (CECH) in response to exposure to pathogens, including arboviruses [36,37,[51][52][53]. Comparison of the transcriptome profiles showed that immunity related genes, including cecropin anti-microbial peptides (CECH) (AAEL000621-RA, AAEL004223-RA, and AAEL017211-RA) were significantly upregulated in the Key West strain Ae. aegypti at two time points (3 h and 3 days) post infection with CHIKV compared with Control of the same strain, and also significantly upregulated in the Orlando strain Ae. aegypti 3 days post infection with CHIKV compared with Control of the same strain. However, AaeCECH was significantly downregulated in the Key West strain Ae. aegypti 3 days post infection with CHIKV compared with the Orlando strain Ae. aegypti (Table 5). AaeCECH was not significantly downregulated in the Key West strain Ae. aegypti 3 days Control compared with Control in the Orlando strain (Table 6). Transcriptomic analysis and q-PCR data showed that AaeCECH gene were highly expressed in the Key West strain and the Orlando strain in response to the CHIKV infection (Tables 2-4, Figure 2C,D). These data may implicate that AaeCECH genes are important for CHIKV infection response, and by extension other arboviruses, in the mosquitoes.
The data presented here represent the first transcriptomic analysis of immune-related genes from a field population of Ae. aegypti and provide useful information for future investigations that aim to elucidate interactions between mosquito vector and arboviruses. Host-virus interactions are representative of complex coevolved adaptations. These dynamic interactions are convincingly demonstrated in the activity of transcriptomes in mosquito hemocytes that comprise the immune system in invertebrates. Designing primers and further examining the innate immune-related genes will provide more information underlying antiviral activity in mosquitoes that limit viral infection. Understanding the mechanism of mosquito-virus interactions, including antiviral defense, and identifying the immune evasion strategies of pathogens will aid in the development of methods to interrupt transmission of arboviruses through the manipulation of mosquitoes.

Aedes aegypti
Two lines of Ae. aegypti from Florida were used in this study. A wild-type line (strain) of Ae. aegypti originated from larvae collected in Key West (24.

Chikungunya Virus Infection
Virus stocks of Chikungunya from Réunion Island (Indian Ocean lineage, LR2006-OPY1, GenBank accession: KT449801) were cultured at 37 • C and 5% carbon dioxide in Vero (African green monkey kidney) cells with media (M199 medium supplemented with 10% fetal bovine serum, penicillin/streptomycin, and myostatin) for an incubation of three days. Following incubation, supernatants from infected cell lines were collected and combined with defibrinated bovine blood and ATP (0.005 M) and presented to four-day-old female mosquitoes for oral feeding using a membrane system (Hemotek, Lancashire, UK). Control blood meals were prepared similarly except that monolayers of Vero cells were inoculated with media only. Samples of infected blood were taken at the time of the feedings for determination of virus titer. Mosquitoes were fed 8.0-8.3 log10 pfu/mL of CHIKV. Specific procedures are described in previous studies [29,35,39].
Blood-engorged mosquitoes were sorted using light microscopy (10×) and transferred to cages (h by d: 10 cm by 10 cm) and incubated at 30 • C and a 12 h light/dark cycle. During incubation, mosquitoes were allowed continuous access to a 10% sucrose solution on cotton pads. Each cohort of mosquitoes in the cages were provided with an oviposition substrate. Mosquitoes 3, 24, 48, 72, 120, 168, and 240 h post feeding were stored at −80 • C for later testing. Separate cohorts of mosquitoes were tested for infection and viral titer on 3, 7, and 10 days following ingestion of CHIKV-infected blood.

RNA Extraction
Samples (10 mosquitoes pool for each sample and three replicates for each time point) were homogenized with a plastic pestle in a 1.5 mL tubes. Total RNAs were extracted using the TRIzol reagent according to the manufacturer's instruction (Ambion, Life Technologies, Carlsbad, CA, USA) and followed the standard protocol [35,39]. The RNA samples were digested by DNase I (RNase-free), according to the manufacturer's instructions (Thermo Scientific, Wilmington, DE, USA). The purified RNA samples were quantitated by NANODROP 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

RNA-Sequencing Library Construction and Sequencing
The RNA-sequencing libraries were constructed at ICBR Gene Expression and Genotyping using NEBNext®Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA), following manufacturer's recommendations. Basically, 500 ng of high-quality total RNA (RIN ≥ 7) was used for mRNA isolation using NEBNext Ploy(A) mRNA Magnetic Isolation module (New England Biolabs, Ipswich, MA, USA). This was then followed by RNA library construction with NEBNext Ultra II Directional Lib Prep (New England Biolabs, Ipswich, MA, USA), according to the manufacturer's user guide. Briefly, RNA is fragmented in NEBNext First Strand Synthesis Buffer by heating at 94 • C for the desired time. This step is followed by first strand cDNA synthesis using reverse transcriptase and oligo dT primers. Synthesis of ds cDNA is performed using the 2nd strand master mix provided in the kit, followed by end-repair and adaptor ligation. Finally, the library is enriched (each library has a unique barcode) by 11 cycles of amplification, and purified by Agencourt AMPure beads (Beckman Coulter, Atlanta, GA, USA, catalog # A63881). Finally, each 14 individual libraries were pooled (total of 3 pools) with equimolar and sequenced by Illumina HiSeq 3000 2X 100 cycles run for total of 1 run (Illumina Inc., San Diego, CA, USA).

RNA-sequencing Data Analysis
Raw reads generated by the Illumina Hiseq 3000 were processed with the Cutadapt (Martin 2011). All partial sequencing adaptors, low-quality bases (phred-like score < 20), short reads (<40 bases), and potential errors from sequencing and library construction were trimmed off or removed. The processed paired-end reads were mapped against the 18,840 transcripts of Ae. aegypti from the VectorBase (https://www.vectorbase.org/organisms/aedes-aegypti/liverpool) by using the bowtie2 mapper (v. 2.3.4.3). The mapping results were further analyzed with samtools and the scripts developed in house at ICBR to remove potential PCR duplicates. Principal component analysis (PCA) was used for quality control, identifying problems with experimental design, mislabeled samples, and to visualize variation between expression analysis samples. The gene expression levels were assessed by counting the number of mapped reads for each transcript [54]. Significantly up-and down-regulated genes were selected using the adjusted p-value, and fold-change for downstream analysis.

Assignments of Gene Ontology (GO) Terms and Pathway Analyses
The GO enrichment analysis (http://amigo.geneontology.org/amigo) were based on the GO terms of Ae. aegypti genes from the VectorBase. The selected genes (p-adj ≤ 0.01) were grouped as the downregulated and upregulated gene pools based on the log2 transformed-fold-change of the RNA-sequencing results, and then assigned to the GO hierarchies and functional categories, including immune system process (GO:0002376), binding (GO:0005488), catalytic activity (GO:0003824), cellular process (GO:0009987), metabolic process (GO:0008152), response to stimulus (GO:0050896), regulation of biological process (GO:0050789), structural molecular activity (GO:0005198), transporter activity (GO:0005215), developmental process (GO:0032502), and signal transducer activity (GO:0004871). The genes that were not assigned GO terms or categorized to other functional groups were assigned as the unknown group.

cDNA Synthesized and qPCR Amplification
cDNAs from 2 µg of total purified RNA were synthesized using a Cloned AMV First-Strand cDNA Synthesis Kit Invitrogen™ and Oligo (dT)20 primer, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). The reaction was terminated by heat inactivation at 95 • C for 5 min. The cDNA samples for qPCR from infected treatment and controls were diluted by adding 80 µL ddH 2 O to 20 µL reaction solution [55,56].
The quantitative PCR (qPCR) assay for target genes AaeDEFA, AaeDEFD, AaeDEFa, AeaCECH, AaeTEP3, and AaeDNR1, and reference gene AaeActin in Ae. aegypti was accomplished using a BIO-RAD C1000 Touch Thermal Cycler, CFX 96™ Real-Time System (BIO-RAD, Hercules, CA, USA). The qPCR reaction mixture with a volume of 15 µL in a Multiwell Plates 96 contained 1 µL diluted cDNA, 0.5 µM primers and 1X master mix of PowerUP SYBR®Green Master Mix (Applied Biosystems, Thermo Fisher Scientific, Foster City, CA, USA). In every qPCR run, AaeActin was employed as an internal control to normalize for variation in the amount of cDNA template. The PCR primers for AaeDEFA, and other genes were designed from the coding region based on GenBank, Accession Number using Primer3 (http://primer3.ut.ee) ( Table 8). The qPCR thermal cycling parameters were the same as in the previous publication [56]. Relative expression levels were calculated as follows for the developmental stages. First, AaeDEFA transcript levels relative to a standard (AaeActin) were calculated using the formula ∆CT = CT (AaeDEFA) − CT (AaeActin). Then, ∆∆CT= ∆CT (infected) − ∆CT (control) value for each sample was calculated. Third, relative expression levels were calculated using the equation 1 × 2 [−average∆∆CT] [35,[56][57][58][59].