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Methodology The study design was based on a before-and-after citywide assessment of Aedes aegypti larval indices and the reported incidence of dengue in Clorinda, northeastern Argentina, over 2003–2007. Interventions were mainly based on focal treatment with larvicides of every mosquito developmental site every four months (14 cycles), combined with limited source reduction efforts and ultra-low-volume insecticide spraying during emergency operations.

The program conducted 120,000 house searches for mosquito developmental sites and 37,000 larvicide applications. Principal Findings Random-effects regression models showed that Breteau indices declined significantly in nearly all focal cycles compared to pre-intervention indices clustered by neighborhood, after allowing for lagged effects of temperature and rainfall, baseline Breteau index, and surveillance coverage. Significant heterogeneity between neighborhoods was revealed. Larval indices seldom fell to 0 shortly after interventions at the same blocks. Large water-storage containers were the most abundant and likely to be infested.

The reported incidence of dengue cases declined from 10.4 per 10,000 in 2000 (by DEN-1) to 0 from 2001 to 2006, and then rose to 4.5 cases per 10,000 in 2007 (by DEN-3). In neighboring Paraguay, the reported incidence of dengue in 2007 was 30.6 times higher than that in Clorinda. Conclusions Control interventions exerted significant impacts on larval indices but failed to keep them below target levels during every summer, achieved sustained community acceptance, most likely prevented new dengue outbreaks over 2003–2006, and limited to a large degree the 2007 outbreak. For further improvement, a shift is needed towards a multifaceted program with intensified coverage and source reduction efforts, lids or insecticide-treated covers to water-storage containers, and a broad social participation aiming at long-term sustainability. Dengue has become the most important viral disease of humans transmitted by arthropods in tropical and subtropical urban regions. Most countries have not been able to maintain permanent larval mosquito control programs against Aedes aegypti, the main vector of dengue and yellow fever, partly because of misguided reliance on using insecticidal space sprays to kill adult mosquitoes. Control actions targeting larvae are based on regular treatment with insecticides of every mosquito developmental site, usually artificial containers found inside or around human dwellings.

The reasons why sustained larval control programs cannot reduce infestations to the desired levels have rarely been investigated. A five-year intervention program conducted in Clorinda, northeastern Argentina, reduced significantly larval infestations compared to pre-intervention levels. Infestations depended on weather variations, pre-intervention larval infestation, and the percentage of houses that were visited and treated with larvicides.

Although the program did not reach the low levels of infestation desired, it most likely prevented or limited new local dengue outbreaks. Large containers for permanent water storage were the most important mosquito development site. For further improvements, a multifaceted intervention program is needed. It should include intensified surveillance and treatment coverage with larvicides that last longer, more efforts on reducing the potential number of mosquito development sites, and a broad social participation aiming at long-term sustainability.

Background data Dengue is a reportable disease in Argentina. Prior to 2000, dengue virus activity was limited in Clorinda (i.e., 36 imported cases in 1999). In February–May 2000 Clorinda experienced a classic-dengue outbreak with 529 suspected and 74 serologically confirmed cases. Most cases were 15–49 years of age, and one-third of them reported travel history to Paraguay before the onset of symptoms. In immediate response to this outbreak, febrile syndromic surveillance commenced at the public hospitals and health care centers, and emergency vector control operations (focal treatment with 1% temephos, indoor space spraying with pyrethroid insecticides, citywide vehicle-mounted ULV insecticide spraying, and elimination of discarded containers in the least affluent neighborhoods) were initiated.

Vector suppressive actions only covered 5% of Clorinda during the second semester of 2001, when the overall prevalence of IgG antibodies to dengue virus was 11.9% (Elena Pedroni, unpublished data, December 2001). Estimates of house and Breteau indices were 22% and 54, respectively. Emergency vector control operations over three weeks reduced house index only by ~45% shortly after interventions. Study design In collaboration with the Municipality of Clorinda, the Ministry of Health of Argentina and researchers from Centro de Investigaciones de Plagas e Insecticidas (CIPEIN), FMS launched a citywide control program in late 2002 following general guidelines for dengue control programs.

Originated in the yellow fever tradition, the desired control targets were a house index. Preliminary survey To establish preliminary infestation levels in domestic and peridomestic habitats and collect information to guide subsequent control operations, a random house survey of 1,808 occupied houses was conducted between November 18 and December 13, 2002. Two-person teams inspected each house for water-filled containers infested with larvae or pupae of Ae. Larval samples from each positive container were collected using ladles and pipettes, placed in test tubes, identified and recorded in household visit forms. Focal cycles A total of 14 cycles of focal treatment aiming at complete coverage was conducted at almost four-month intervals (mean, 115 days; standard deviation, SD, 29 days) from March 2003 to December 2007 (). An average of 33 (SD, 7) people participated in each cycle.

The total local labor effort over the five-year period was 1,820 person-months. In addition to the container inspections described in the preliminary surveys, field teams also (1) removed or emptied disposable containers (manual treatment); (2) encouraged householders to manage containers on their property, and (3) treated water-storage containers (non-drinking water) with appropriate 1% temephos in sand granules at 1 mg per liter, or less frequently, with Bacillus thuringiensis israelensis (BTI, VectobacR, Bayer) applied as rings of prolonged residual power. Field teams recorded the number of residents and grams of larvicide applied in each house, but did not record the number of containers treated manually. Inspection and larviciding efforts in cemeteries and used tire lots were conducted during each focal cycle. Coverage was mapped and continuously updated so that multiple attempts could be made to gain access to as many homes as possible.

Occupied houses that could not be inspected on the first visit were re-visited at the end of each focal treatment cycle to enhance surveillance coverage. At the community level, educational efforts were supplemented by nine community workshops conducted in schools distributed throughout the city (one cycle only at the first cycle).

Summary of interventions and larval indices at the preliminary survey and over focal treatment cycles 1–14 in Clorinda, Argentina, 2002–2007. During focal cycles 1–7, the presence of larvae and pupae was recorded on datasheets only, whereas in cycles 8–14 samples of larvae and pupae from each positive container were transported to the laboratory where they were identified to species after development to the adult stage. Immature stages in positive containers nearly always were Ae. Additionally, starting on early 2006 (focal cycle 10), containers were classified into eight mutually exclusive categories: A, tires; B, tanks, barrels and drums; C, flower vases; D, construction materials and discarded vehicle parts; E, bottles, cans and plastic goods; F, wells; G, natural containers; and H, other types (mainly ceramic pots, tin cans, pieces of canvas, canvas pools and broken or unused appliances such as refrigerators or washing machines) so that the most productive container types could be identified. The focal treatment cycle 10 (started in mid-February 2006) was interrupted due to an emergency response to a region-wide dengue outbreak. Supplementary indoor and city-wide truck-based ULV spraying campaigns were carried out in March–May 2003, August–September 2004, March–May 2006, and February–May 2007; application dates and interventions specific details are summarized in and. Evaluation surveys From 2003 to 2005 (cycles 1–7), selected teams among those conducting regular control operations assessed the impact of larviciding operations shortly after them (mean = 41.8 days; 95% confidence interval, 37.4–46.3) in a convenience sample of blocks distributed throughout the city (i.e., early post-intervention assessment surveys).

The total number of houses inspected for early infestation was 5,933 and averaged 448 houses per cycle (SD, 411). This additional monitoring scheme did not include additional treatments and was subsequently phased out because it lengthened the duration of focal treatment cycles. Data analysis The data collected over all cycles of focal treatment, monitoring and indoor house sprays were entered in an Access R database to calculate the outcome parameters: the house index (i.e., the percentage of inspected houses that were positive for Ae. Aegypti larvae or pupae); the Breteau index (i.e.

The number of water-holding containers that were positive for Ae. Aegypti larvae or pupae per 100 houses inspected); the percentage of all visited houses that were closed or vacant or whose occupants were unavailable on first visit; the percentage of all visited households who denied entry to premises, and of all visited houses that were treated with larvicides (temephos or BTI) in each cycle. The first and third quartiles of the citywide house and Breteau index were calculated with package Hmisc in R software; quartiles were based on the appropriate larval index value at each neighborhood j weighted by the number of houses inspected at each j and focal cycle i (i.e., time post-intervention, range 1–14). Breteau and house indices were checked for normality and then transformed to logarithms to the base 10 of count+1.

Two-tailed paired t tests were used to examine differences between larval indices recorded in the preliminary survey and the first focal treatment cycle at each neighborhood. Odds ratios (OR) and incidence rate ratios (IRR) were estimated to assess the effect size of control interventions at each neighborhood j and cycle i compared with pre-intervention (baseline) values at each j and i = 1 by fitting random-effects regression models clustered by neighborhood to time-dependent larval indices using Stata 9.0. The use of random-effects models responds to the fact that households within a neighborhood roughly share the same environment and other undetermined characteristics that can create dependencies between responses (positive containers or houses) within the same geographic unit. The outcome measures were the Breteau and house indices at each j and cycle i.

The main model included as explanatory variables the effects of focal cycle number (a categorical variable that compares each cycle 2–14 to the pre-intervention cycle 1); temperature and rainfall at different time lags before the exact onset of cycle i at j (see selection procedures below); the intensity of surveillance coverage at j and i−1 (i.e., a continuous variable measured by the proportion of total houses at j that were inspected at the immediately preceding focal cycle), and the corresponding baseline larval indices at j and i = 1 (pre-intervention). Pre-intervention larval indices were included as predictors based on the notion that the most infested neighborhoods before the intervention program may continue having more foci after interventions. The entire set of predictors was selected a priori based on existing knowledge. Variations in house and Breteau indices were tested with the commands glm (family binomial) and xtmixed using maximum likelihood procedures in Stata 9.1. The short-term effects of interventions on larval indices (as determined by the early post-intervention surveys at cycles 1–7) at fixed blocks within infested neighborhoods were assessed using the same procedures described before for each cycle separately. A dummy variable measured the effects of interventions at each cycle, and a time lag of 7 days was used for temperature and rainfall in all cycles. Selection of the most adequate time lag and variables for representing weather effects was based on Akaike Information Criterion (AIC).

Four sets of candidate variables were identified: mean daily temperature; minimum daily temperature; maximum daily temperature, and daily rainfall averaged for each neighborhood at every focal cycle. Each of these variables was computed for several weekly time lags (range, 0–7 weeks). The set of temperature variables was considered linearly or by adding a quadratic term to account for putative non-linear effects. For each set of weather-related variables, models including non-weather variables were fitted to Breteau indices for each time lag. The best lag was identified by the model with the smallest AIC. Models with every combination of the four selected lagged variables were computed in order to select the best model including all the variables considered. Results Mean house (19.5%) and Breteau indices (22.5) at the preliminary survey in late 2002 were consistent with those recorded before interventions in late 2001 (22% and 54, respectively).

Neighborhood-specific Breteau indices decreased between the preliminary survey and the first focal cycle before larviciding operations but the difference was not statistically significant (t = 1.59, df = 33, P = 0.122). In contrast, the observed decrease in neighborhood-specific house indices was significant (t = 2.42, df = 33, P = 0.021). Mosquito larvae or pupae collected during surveys were predominantly Ae.

Aegypti; only Culex sp. 2007 Ford Focus Driver Door Panel Removal. (but no Aedes albopictus) were detected in a few containers. House and Breteau indices recorded at the first focal cycle varied widely between neighborhoods (coefficient of variation, 70% and 89%, respectively). The distribution of Breteau indices was bimodal and highly skewed, with 10 neighborhoods exceeding 30. Between late 2002 and 2007, ≈170,000 households were visited and of these, 120,000 were surveyed for Ae. Aegypti and 37,000 were treated with larvicides (). Household inspections per cycle averaged 8,511 (SD, 2,127; range, 5,587 to 12,366) ( and ).

On average, 25% (SD = 2%) of the houses visited in each cycle were closed or vacant or with householders temporarily absent on first visit, and 3% (SD = 1%) denied entry for inspection of premises (Figure S2). The denial rate declined from 7% at baseline to 3% at 21 months post-initial intervention (MPI) (focal cycle 6, November 2004) but increased again to 5% at 29–33 MPI (cycles 8–9, June–October 2005), only to decline again to 1–2% at 41–47 MPI (cycles 12–14, August 2006–June 2007) when a dengue outbreak emerged in Paraguay in early 2006.

The percentage of visited houses that were treated with larvicides averaged 22.2% (SD = 2.8%). The ratio between larvicide-treated houses and Ae. Aegypti positive-houses averaged 5.8 (SD = 3.1) but varied widely from 2.8 to 13.5 over time.

The total number of positive containers detected (mean = 738, SD = 418) and the kilograms of temephos applied (mean = 193 kg, SD = 45) at each focal cycle were not significantly correlated (r = 0.350, n = 14, P>0.1). Citywide house indices declined sharply from 13.7% at baseline to 3.7% at the second focal cycle conducted mostly through spring of 2003 whereas Breteau indices declined from 19.0 to 4. Access Serial Port Windows 7. 8 (). Larval indices then fluctuated seasonally and peaked every year between summer and early fall, with large variations between neighborhoods within anyone focal cycle as expressed by interquartile ranges.

Monthly house and Breteau indices at a citywide scale over the five years were highly positively correlated (r = 0.966, n = 60, P. Distribution of mean house and Breteau indices, percent of houses treated with larvicides, and control interventions in Clorinda, Argentina, 2003–2007. Weather-related variables exerted highly significant effects on larval indices, especially when time lags were allowed for (). Based on AIC scores, the best lags were 1 week for mean daily rainfall and mean minimum daily temperature, and 4 weeks for mean daily temperature and mean maximum daily temperature. In every case the best lag identified clearly surpassed other candidate lags (range of ΔAIC of weather variables, 5.6–11.0).

Because substantial model selection uncertainty was found when comparing models including temperatures with and without quadratic terms, the linear variables were selected for the remaining analyses. Using the selected time lags for each variable, the best model found included mean rainfall, mean temperature and mean minimum temperature. All of the selected variables had a positive association with larval indices (). Random-effects multiple regression models of the effects of focal treatment cycles on house indices (logistic model) and Breteau indices (linear model) relative to cycle 1 in Clorinda, Argentina, 2003–2007. Control actions exerted significant impacts on larval indices, with exception of a limited upsurge at 36 MPI (cycle 10, February 2006) ().

When post-intervention larval indices at focal cycles 2–14 were compared to pre-intervention indices at cycle 1 by random-effects multiple regression, log-transformed Breteau indices declined significantly (P0.3). Breteau indices showed similar patterns.

Short-term impact of larviciding and manual operations on larval indices at the same blocks in focal treatment cycles 1–7 in Clorinda, Argentina, March 2003–April 2005. The abundance and infestation of water-holding container types and distribution of larval indices differed largely among types of container. For example, at focal cycle 12 (spring–summer 2006–2007), tanks, barrels and drums for water storage (B type) were the most abundant containers (4,380) and the most likely to be infested (15.2%), accounting for 49% of all infested containers found (). The second most important container class was disposable bottles, cans and plastics (E type), which were abundant (1,476) and as frequently infested (15.0%) as B containers, but accounted for only 16% of all infested containers. The frequency distribution of containers per type in cycle 12 was highly overdispersed between neighborhoods, with coefficient of variations that increased from 36% (type B) to 288% (D). Incomplete surveillance coverage. Vector surveillance and insecticide treatment coverage were high but incomplete in space and time despite the community's high levels of acceptance of suppressive measures.

House indices were inversely related to surveillance coverage. Houses lost-to-inspection may have constituted a persistent source of infestations of unknown magnitude. Re-visiting houses at appropriately scheduled times increased surveillance coverage from late 2006 forward. Despite its importance elsewhere, intensified searches for alternative developmental sites in non-residential habitats (tree holes and bromeliads) yielded negative results, and only 3.4% of indoor containers were found to be infested in early fall 2007 (unpublished results); •. Limited residuality of temephos.

The expected duration of temephos residual effects (about three months) and standard control guidelines, further compounded with operational restrictions, dictated that each focal treatment cycle took an average of almost four months. However, the residuality of temephos has recently been shown to vary widely between. Permanent developmental sites. In the absence of major changes in the management of large containers for permanent water storage, these mosquito developmental sites were continuously available, rainfall-independent, and varied little in frequency among neighborhoods. Unfortunately, the intervention program did not seek to identify the most productive containers for targeted control, and the intervention protocol only suffered minor adjustments over time. Pupal surveys conducted in a large neighborhood in 2007 showed that large tanks used for potable water storage were both the most abundant and the most productive type of container; •. Suitable climatic conditions.

The prevailing local weather conditions are very favorable for Ae. Aegypti (as shown by the presence of immature stages on every month over the five years), and would determine fast egg-to-adult developmental times ranging from nearly 6 days in summer up to 10 days in early spring or fall according to experimental data.

The gonotrophic cycle length of female Ae. Aegypti would have been 3.8 days at local mean temperatures (27°C) over January–March 2003–2007, as interpolated from tables in. Larval indices were highly significantly associated with average minimum temperatures and rainfall one week earlier and average temperatures 4 weeks earlier. These data suggest that recent cool temperatures and rainfall influenced variations in Ae. Aegypti larval indices, as other studies also showed for female and larval abundance. Fast development rates and the limited residuality of temephos during hot weather jointly create a window of opportunity in which the vector population recovers partially between focal cycles and then spreads by flight dispersal at a local scale; •.

The triatomine bug Rhodnius prolixus is a main vector of Chagas disease, which affects several million people, mostly in Latin-America. Host searching, pheromone communication, and microclimatic preferences are aspects of its behaviour that depend on multimodal sensory inputs. The molecular bases of these sensory processes are largely unknown. The expression levels of genes transcribed in antennae were compared between 5 th instar larvae, and female and male adults by means of RNA-Seq. The antennae of R. Prolixus showed increased expression of several chemosensory-related genes in imaginal bugs, while both sexes had similar expression patterns for most target genes.

Few cases suggest involvement of target genes in sexually dimorphic functions. Most odorant and ionotropic receptor genes seemed to be expressed in all libraries. OBPs and CSPs showed very high expression levels. Other sensory-related genes such as TRPs, PPKs and mechanoreceptors had consistent levels of expression in all libraries. Our study characterises most of the sensory gene repertoire of these insects, opening an avenue for functional genetics studies.

The increase in expression of chemosensory genes suggests an enhanced role in adult bugs. This knowledge allows developing new behaviour interfering strategies, increasing the options for translational research in the vector control field. Transcriptome assemblies The sequencing yield was 353.6 M read-pairs, distributed as 103 M for larval, 125 M for female, and 125.6 M for male libraries. A total of 128,047 contigs, with an average length of 744 bp, were assembled using SOAPdenovo. The length of contigs ranged from 100 to 35,978 bp and N50 value was 2,518. Using Trinity, a total of 303,403 contigs with an average length of 368 bp were assembled.

The length of contigs ranged from 101 to 29,400 bp and N50 value was 649. Gene Ontology annotation and functional enrichment analysis A total of 239; 249 and 177 transcripts had FPKM values >1,000 in larvae, female and male antennal transcriptomes, respectively.

After the annotation of these transcripts based on Gene Ontology terms, a functional enrichment analysis was performed for each antennal library comparing it to the R. Prolixus genome. Those transcripts enriched in the antennal transcriptomes and their Gene Ontology annotations are detailed in. The number of genes that shared a Gene Ontology annotation term in each library and those that were unique are detailed in. The following terms deserve to be highlighted: GO:0005513 (detection of calcium ion); GO:0010880 (regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum); GO:0033173 (calcineurin-NFAT signalling cascade); and GO:0060316 (positive regulation of ryanodine-sensitive calcium-release channel activity) that were common to all libraries ().

Triggering Ca 2+ flux activates a number of signalling pathways including but not restricted to nucleoside diphosphate kinase activity and nucleoside diphosphate phosphorylation, which appeared only in the male library (GO:0004550 and GO:0006165, respectively in ). This finding suggests the existence of signalling pathways that are unique for each library and that should be experimentally explored in further work. The term GO:0005549 (odorant binding) was shared by all libraries (). Additionally the ability to perceive environmental light is important for the normal growth and development of many organisms and the GO:0009785 (blue light signalling pathway) was also shared among larval, female and male libraries (). Manual gene curation The transcript sequences allowed us to manually improve the gene models for 16 ORs, two GRs, 12 IRs, seven OBPs, three CSPs, two ammonium transporters, four TRPs, two PPKs, and Piezo and narrow abdomen genes.

In addition two IRs identified as pseudogenes in the genome ( Ir75d and Ir75g with VectorBase codes RPRC000105 and RPRC017356, respectively) had coding sequences in the antennal transcriptome that allowed their modelling as intact genes. Additional sensory genes were identified in the R. Prolixus genome by tBLASTn searches against the VectorBase database using orthologous sequences from other insects. The sequences of these genes were subsequently compared to our transcriptome assemblies and corrected or extended.

Five CheB protein sequences were identified in the R. Prolixus genome (VectorBase codes: RPRC004662, RPRC004663, RPRC004664, RPRC004665 and RPRC004666) and annotated as CheB1- CheB5 respectively (). The sequence of CheB1 (RPRC004662) was manually edited. No sequence belonging to the CheA protein family was found in the bug genome. Only one SNMP sequence (RPRC013907) had been annotated in the bug genome, and our new searches of the genome identified four additional ones: Snmp1b (RPRC013910); Snmp1c (RPRC000399); Snmp1d (RPRC002720); and Snmp2 (RPRC002754) (). Several other sequences discovered through tBLASTn searches against the VectorBase had to be manually edited according to our transcriptome assemblies. Another 13 members of the SCRB/CD36 protein family were identified and 9 of their models improved ().

A new ammonium transporter was identified: Amt1 (RPRC006389). The previously identified ammonium transporter ( Amt) was now annotated as Amt3 according to tBLASTn results.

The sequences of Rh50 and both Amts were manually edited after comparing them to our transcriptome assemblies. In the case of TRP receptor genes, six new members and a new TRPA1 isoform were identified and annotated ( TrpA5a; TrpA5b; TrpA5c; Pkd2; TRPM; and TRPML), raising the number of R. Prolixus TRPs to 14.

The sequence described as pyrexia in the genome paper (identified as RPRC000570) was properly annotated as TRPA5a based on its sequence similarity to other TRPA5 members. Additionally, the sequence of TRPML needed to be manually edited after comparison with the originally described gene. Four new PPK receptors were identified, bringing the total of R. Prolixus PPKs to ten. Finally, new orthologous sequences of known insect mechanoreceptor genes were identified: mrityu (RPRC014507), two type B chloride channels (RPRC0065358 and RPRC013530), and three NMDA receptors (RPRC000296; RPRC006099; and RPRC001831). The sequences of mirtyu and both chloride channel-b genes were manually edited after comparison to our transcriptome assemblies.

The nucleotide sequences of all genes analysed in this work are included in, and their VectorBase predicted protein codes and their functional annotations are detailed in. An edited version of the R. Prolixus genome generic file format (GFF) was created and all the adjustments included (see ). Mapping and transcript prediction The Illumina reads were mapped to the modified version of the RproC1 R.

Prolixus genome assembly (see Material and Methods section) to analyse gene expression profiles. The BioProject PRJNA281760 in the Sequence Read Archive at NCBI contains all the RNA-Seq reads produced in this study. A high proportion of reads mapped to the R. Prolixus genome, being 92% for larval, 94.6% for female and 94.3% for male libraries. A total of 17,190 genes and 17,353 isoforms were predicted by Cufflinks-Cuffmerge in the consensus transcriptome based on the three conditions studied. Pair-wise transcriptome comparisons The expression levels of all transcripts (represented as Log10 of FPKM +1) were compared in pair-wise regression analyses and visualized using scatter plots ().

The comparisons between larval vs. Female (, regression slope coefficient = 0.9612, R 2 = 0.903) and larval vs. Male (, regression slope coefficient = 0.986, R 2 = 0.872) antennal transcriptomes showed an overall similar gene expression for antennae of larvae and adults.

Regarding male and female antennae, both transcriptomes showed a similar global expression profile (, regression slope coefficient = 0.909, R 2 = 0.918). Interestingly, several transcripts showed higher expression in the male than in the female transcriptome (those with Log10 FPKM +1 lower than 2.5, ). On the left, comparison of expression levels of the 17,190 genes predicted: between larval vs. Adult female transcriptomes ( a); larval vs. Adult male transcriptomes ( b) and; female vs.

Male adult transcriptomes ( c). On the right, comparisons of the expression levels of a set of 217 sensory receptor genes between the larval vs.

Adult female transcriptomes ( d); larval vs. Adult male transcriptomes ( e) and; between female vs. Male adult transcriptomes ( f). Dotted lines indicate 1:1 gene expression relationship between the compared antennal transcriptomes, while linear regression analyses were marked with blue solid lines. QRT-PCR validation of RNA-Seq The expression levels of 25 genes and two reference genes () were evaluated by means of qRT-PCR to validate these RNA-Seq expression data.

Transcript abundances obtained through both techniques were strongly correlated when larval vs. Male results (Spearman Correlation; r = 0.88, p 1 in larval antennae, a similar proportion of genes seem to have increased expression in adult antennae (see and ). Consistently, statistical analysis based on the edgeR package showed that while 6 ORs increased their antennal expression significantly in both sexes after the imaginal moult (), five ( Or18; Or33; Or40; Or54; and Or58) did so exclusively in female antennae and, six ( Orco; Or39; Or46; Or62; Or84; and Or109) augmented only in male antennae (). Considering all these cases (all belonging to the highest fold-change category in ), almost a sixth of R. Prolixus ORs had a significant increase in their expression in the antennae of adult bugs (FDR adjusted p-value. The OR evolutionary history was inferred using the Maximum Likelihood method in MEGA6.0.

The bootstrap consensus tree (which topology is displayed) was inferred from 1,000 pseudo-replicates. Only bootstrap values higher than 70 are displayed. An initial tree was obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using JTT and F models. Expression levels (represented as Log10 FPKM +1) are depicted with a colour scale in which white and orange represent lowest and highest expression, respectively. Suffixes to gene names are explained in the Methods section.

Ionotropic receptors Considering our different expression criteria, almost all IR receptors seem to be expressed in bug antennae (). As seen for ORs, many IRs seem to have increased expression in the antennae of adult bugs (). A similar increase was observed for the three IR co-receptors, reinforcing the apparent effect of imaginal molt on IR gene expression.

Interestingly, the most expressed IR gene was Ir75a (7.5, 43, and 39 FPKM in larvae, female and male antennae, respectively), and not the IR co-receptor genes, which showed a maximum FPKM of 13 (). A subset of IRs ( Ir41c; Ir40; Ir93a; Ir106; Ir75g; and Ir75o) showed expression profiles similar to that of Ir25a (). Co-receptors Ir8a and Ir76b showed similar expression profiles, with apparent increases in male antennae ( and ). Furthermore, 11 out of 16 genes belonging to the IR75 expansion also seem to have higher expression in male antennae ( and ).

The IR evolutionary history was inferred using the Maximum Likelihood method in MEGA6.0. The bootstrap consensus tree (which topology is displayed) was inferred from 1,000 pseudo-replicates. Only bootstrap values higher than 70 were displayed. Initial tree was obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the WAG and F models. Expression levels (represented as Log10 FPKM +1) were depicted with a colour scale in which white and orange represent lowest and highest expression, respectively.

Suffixes to gene names are explained in the Methods section. Almost a third of the IR receptor genes do not seem to be expressed in larval antennae (FPKM value 1 (), a third of which seem to have increased transcript abundance in male antennae (). Gustatory receptors BLASTn searches against our transcriptome assemblies suggest that only nine GRs were expressed in the antennae. Shows that this number can rise to 20 depending on the criteria used. The level of antennal expression seemed to relate to gene distribution in the phylogenetic tree ().

The expression of Gr26, Gr27, and Gr28 was relatively high () in all conditions studied (). The genes Gr1 (orthologue of D. Melanogaster fructose receptor), Gr2, Gr20 and Gr24 seemed to have increased expression in the antennae of adult bugs (,). Most GRs (22/28) showed FPKM values. The GR evolutionary history was inferred using the Maximum Likelihood method in MEGA6.0. The bootstrap consensus tree (which topology is displayed) was inferred from 1,000 pseudo-replicates. Only bootstrap values higher than 70 were displayed.

Initial tree was obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using JTT and F models. Expression levels (represented as Log10 FPKM +1) were depicted with a colour scale in which white and orange represent lowest and highest expression, respectively. FX: gene model corrected based on the de novo transcriptome assembly. Odorant binding proteins and chemosensory proteins A total of 27 OBPs and 19 CSPs have been annotated in the R. Prolixus genome and most of them were expressed in bug antennae (). As expected from studies in other insects, most OBPs and CSPs showed very high levels of expression (), up to 100X higher than Orco, the most highly expressed odorant receptor.

Expression patterns of both protein families seemed to correlate with their clusterization into the corresponding phylogenetic trees (). Many of these genes did not show relevant expression increases after imaginal moult, even though a few of them were more highly expressed in the antennae of adults (). This was the case for Obp12 for which expression was significantly increased in the antennae of males and females (). Besides, Obp6, Obp18 and Obp25 had increased expression in the antennae of female bugs when compared to those of larvae (). A single CSP gene ( Csp11) showed significantly increased expression in the antennae of females when compared to those of larvae (). The OBP and CSP evolutionary histories were inferred using the Maximum Likelihood method in MEGA6.0. The bootstrap consensus trees (which topologies are displayed) were inferred from 1,000 pseudo-replicates.

Only bootstrap values higher than 70 were displayed. Initial trees were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using a LG model. Expression levels (represented as Log10 FPKM +1) were depicted with a colour scale in which white and orange represent lowest and highest expression, respectively. FX: gene model corrected based on the de novo transcriptome assembly. CheB, SNMP/CD36 and ammonium transporter proteins All CheB genes are expressed in the antennae () and interestingly, most of them showed higher expression in female antennae ().

In the case of the SNMP/CD36 protein family, all SNMPs were expressed in antennae (). Snmp1a and Snmp1b showed higher expression in adult antennae, while Snmp2 presented high expression in antennae of all developmental stages studied ( and ). SCRB8a and SCRB9 were the scavenger-like receptors showing highest expression (). The three ammonium transporters were expressed in the antennae ( and ); especially Amt1 with high expression in antennae of all developmental stages studied ().

Phylogenetic relationships of CheB ( a) and sensory neuron membrane protein/CD36 protein ( b) families and their corresponding heat maps. Heat map of ammonium transport proteins ( c). Heat maps compare expression levels in antennae of larvae (L), female (F) and male (M) adults of R. The evolutionary history of CheB and SNMP/CD36 protein families were inferred using the Maximum Likelihood method in MEGA6.0.

The topologies of the CheB (highest log likelihood −1123.0068) and SNMP/CD36 trees (highest log likelihood −6147.2094) are shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The initial trees for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using WAG and F models models (for CheB proteins) and L and G models (for SNMP/CD36).

Expression levels (represented as Log10 FPKM +1) were depicted with a colour scale in which white and orange represent lowest and highest expression, respectively. FX: gene model corrected based on the de novo transcriptome assembly; NTE: N-terminus region is missing. Phylogenetic relationship of transient potential receptor (TRP) genes and their corresponding heat map ( a). Heat map of potential mechanoreceptor genes ( b). Heat maps compare the expression levels in antennae of larvae (L), female (F) and male (M) adults of R. The TRP evolutionary history was inferred using the Maximum Likelihood method in MEGA6.0.

The bootstrap consensus tree (which topology is displayed) was inferred from 1,000 pseudo-replicates. Only bootstrap values higher than 70 were displayed. The initial tree was obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using WAG and F models. Expression levels (represented as Log10 FPKM +1) were depicted with a colour scale in which white and orange represent lowest and highest expression, respectively. FX: gene model corrected based on the de novo transcriptome assembly.

In the case of putative mechanoreceptors, piezo deserves mention as the gene showing the highest expression. Overall, males tended to have higher expression of mechanoreceptor genes compared to females ().

Detoxification enzymes Sixteen CYP4 genes showed FPKM values higher than 1 in at least one library (). Five CYP4 genes presented high transcript abundances in all libraries ().

Besides, several genes belonging to this clade showed higher expression in libraries obtained from adult antennae (). Intriguingly, some members of this clade had higher expression in larval or female antennae. Seven secreted esterase genes presented FPKM values >1 in at least one library ().

Few of these enzymes presented high expression in all libraries, while a small number presented higher expression profiles in the antennae of larvae, adults or females (). The present study represents the first antennal transcriptome sequenced for a Chagas disease vector and one of the few existing for hemimetabolous insects. It improves our characterization of the sensory repertoire of R. Prolixus, which was initially described in the recently published genome paper, allowing more robust functional studies. Antennal transcriptomes of several insect species have been recently published, including those of tree-killing beetles or human disease vectors. Most of these studies have been carried out with holometabolous insects and focused on imaginal antennae. In the case of hemimetabolous insects, antennal transcriptome analyses are fewer and many aspects of the molecular bases of their sensory physiology are still unknown.

One of the main outcomes of this study is that these insects seem to increase the expression of several chemosensory genes after reaching the adult phase, as significantly increased expression was observed for diverse odorant receptor, OBP, CSP and SNMP genes which may serve to enhance adult chemosensory abilities. In contrast, antennal transcriptomes of male and female adults showed similar expression profiles (). A few exceptions showing higher expression in males or females may be related to functions that are differentially relevant for one of the sexes. These genes should be further studied as candidates mediating sexually dimorphic physiological activities, such as pheromone detection. The number of olfactory receptors apparently expressed in the antennae of R. Prolixus (88 ORs and 22 IRs, see ) is much higher than that of glomeruli (22) identified in the antennal lobe of this species. An opposite case has been observed for locusts, suggesting that the prevailing view of “one receptor-to-one glomerulus” olfactory organization scheme may not be generalized, and alternative neuronal organization schemes for the olfactory system may co-exist in the diverse insect clades.

We characterized the antennal expression levels of all known R. Prolixus sensory-related genes and compared their transcript abundances in the antennae of 5 th instar larvae and adults for the first time in a hemimetabolous insect. As said above, increased expression from larvae to adults was observed () for many chemosensory-related genes. Indeed, sexual behaviour, oviposition and flight are activities exclusively performed by adult bugs, and their incorporation into bug biology seems to correlate with these peripheral modifications happening at the molecular level. Consistently, this increase in receptor gene expression also seems to correlate with the greater number of chemosensilla reported for the antennae of adult insects of this species. As observed in other insects,, Orco was the odorant receptor showing highest expression in bug antennae () and the imaginal increase in Orco expression seen in our transcriptome was previously reported for R. Prolixus antennae based on qRT-PCR.

Interestingly, only one OR presented differential expression between sexes (), while the bulk of the chemosensory repertoire seemed to remain mostly similar. A similar case was observed in an antennal transcriptome from the blowfly Calliphora stygia. Additional functional studies, such as RNAi or de-orphanization through heterologous gene expression would be necessary to understand the role of these genes in adult bug biology. In the case of ORs and other sensory receptor genes which presented similar expression in larval and adult antennae, a potential role in host, shelter and aggregation signal detection deserves to be considered in future genetic studies. As seen in the results section, a parallel increase of IR transcript abundance also seemed to occur after imaginal moult (). Given that Ir75a was the most highly expressed IR in adult bug antennae (), even higher than Ir25a, it seems as a potential candidate for functional studies. Interestingly, the antennae of male bugs showed increased expression of some IR co-receptors ( Ir76b and Ir8a), part of the Ir75 gene subfamily, as well as other specific receptor genes like Ir41a and Ir41b ( and ).

Whether the proteins encoded by these genes are related to male-enhanced functions needs to be explored. As it might be expected given their primary role in gustation, most GRs showed low or no expression in bug antennae, in agreement with observations in other insects. It is likely that these receptors are involved in chemoreception in bug tarsi or proboscis. Indeed, triatomines use contact chemo-signals to mark shelters and to recognize sexual partners. Two groups of GRs, nevertheless, showed interesting expression patterns in antennae, these being Gr1 and Gr2 primarily being expressed in adults, and Gr26–28 highly expressed in all stages studied (). These genes may relate to triatomine behaviours known to be based on contact chemostimuli, even though other unexpected roles cannot be discarded. Antennal transcriptome studies performed with other insects showed similar expression patterns for OBPs and CSPs.

Bug OBP and CSP numbers (27 and 19, respectively) are much smaller than those of ORs and IRs (111 and 33, respectively). This suggests that their role as odour carriers is not necessarily linked to all specific receptors. The increase in expression observed for ORs and IRs at the adult stage had a correlate in the case of several odour-carrying proteins, making them potential candidates for mediating adult-related functions. CheB proteins have been related to the detection of cuticular hydrocarbons in Drosophila and as said above, compounds of this type have been shown to mediate bug communication. Nevertheless, no behavioural or functional evidence can be related to date to the increased expression observed for R. Prolixus female bugs (). Two SNMPs with high expression in adults ( Snmp1a and Snmp1b, ) may have relevant roles for imaginal chemosensory physiology.

Snmp1 is important for sex pheromone responses in Drosophila and moths,; however, functional data on other insect models are scarce for this protein family. The high expression of ppk-like10 suggests a relevant role in the sensory ecology of these bugs.

Similarly, special mention should be made for painless and waterwitch genes, the most expressed TRPs (). This expression profile suggests their relation with the known capacity of these bugs to detect heat and water vapour,, as Drosophila painless and waterwitch genes encode for heat and water vapor receptors, respectively.

The relatively high expression of several mechanoreceptors () seems to correspond to the known capacity of triatomines to detected vibratory signals. It is worth highlighting that several genes belonging to this category seemed to have more intense expression in the antennae of male bugs, which are known to detect vibratory sex signals. The homogenous expression profile observed for PPKs and TRPs in larval and adult antennae, differently from ORs and IRs, reinforces the hypothesis of a major role in the detection of salts, substrate-borne vibrations, heat or water vapour, which are mostly relevant for all triatomine developmental stages. Several secreted esterases and CYP4 clade members presented high levels of antennal expression; therefore suggesting that they may be involved in odour/pheromone degradation processes. However, it would be important to compare the expression of these genes in other bug tissues and evaluate the corresponding levels of enzymatic activity to reinforce a potential role as ODEs. The statistical comparison of larval vs.

Female and larval vs. Male RNA-seq data with results obtained by means of qRT-PCR for 25 selected genes demonstrated similar trends for antennal transcript abundances reinforcing the significant increase observed in the expression of several chemosensory genes in bug antennae after imaginal moult. On the contrary, transcript abundances obtained by both techniques showed a low correlation when results from female and male samples were compared. In fact, gene expression differences between sexes detected by means of both techniques were small, i.e. Fold-changes obtained for 22 genes were lower than 0.30.

Coincidently, absence of sexual dimorphism has been reported for the number of antennal chemosensilla and glomeruli in the antennal lobe of R. The similarities in male and female bug chemosensory systems may explain, at least in part, the low correlation observed when results from both techniques were compared.

The number of transcripts predicted in the antennal transcriptome (17,190) is similar to that reported (16,857) in the last version of the R. Prolixus genome (.). More than 10,000 genes were expressed in antennae, meaning that at least 60% of all genome genes were expressed, similarly as seen for D. Melanogaster antennae. Our two de novo transcriptome assemblies and the subsequent manual curation process have allowed validating a large number of gene models, while many additional sensory-related genes have also been identified here. An edited version of the R. Prolixus genome generic file format (GFF) was created including these adjustments (), improving the potential of future RNA-Seq analyses with this insect species.

Accession codes: Read sequences from the three libraries have been submitted to the Sequence Read Archive (SRA) and NCBI under the project accesion number PRJNA281760/SRP057515 and the SRA accession numbers for the three experiments can be accessed at SRS923612/SRX1011796/SRR2001242 (antennal library from larvae); SRS923595/SRX1011769/SRR2001240 (antennal library from female adult); and SRS923599/SRX1011778/SRR2001241 (antennal library from male adult). How to cite this article: Latorre-Estivalis, J. The molecular sensory machinery of a Chagas disease vector: expression changes through imaginal moult and sexually dimorphic features. 7, 40049; doi: 10.1038/srep40049 (2017).

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