Survival rate, blood feeding habits and sibling species composition of Aedes simpsoni complex: Implications for arbovirus transmission risk in East Africa.

Aedes simpsoni complex has a wide distribution in Africa and comprises at least three described sub-species including the yellow fever virus (YFV) vector Ae. bromeliae. To date, the distribution and relative contributions of the sub-species and/or subpopulations including bionomic characteristics in relation to YF transmission dynamics remain poorly studied. In this study conducted in two areas with divergent ecosystems: peri-urban (coastal Rabai) and rural (Rift Valley Kerio Valley) in Kenya, survival rate was estimated by parity in Ae. simpsoni s.l. mosquitoes sampled using CO2-baited BG Sentinel traps. We then applied PCR targeting the nuclear internal transcribed spacer 2 (ITS2), region followed by sequencing and phylogenetic analytics to identify the sibling species in the Ae. simpsoni complex among parous and blood fed cohorts. Our results show that Ae. bromeliae was the most dominant sub-species in both areas, exhibiting high survival rates, human blood-feeding, and potentially, high vectorial capacity for pathogen transmission. We document for the first time the presence of Ae. lilii in Kenya and potentially yet-to-be described species in the complex displaying human feeding tendencies. We also infer a wide host feeding range on rodents, reptile, and domestic livestock besides humans especially for Ae. bromeliae. This feeding trend could likely expose humans to various zoonotic pathogens. Taken together, we highlight the utility of genotype-based analyses to generate precision surveillance data of vector populations for enhanced disease risk prediction and to guide cost-effective interventions (e.g. YF vaccinations).

Introduction

Yellow fever (YF) is a re-emerging arboviral threat in Africa and South America despite availability of an effective vaccine to protect humans. This is unparalleled in eastern Africa exemplified by increased frequency and magnitude of outbreaks recorded recently in Sudan (2012), South Sudan (2020), Uganda (2011, 2016, 2019, 2020), and Ethiopia (2012–2014, 2018; 2020) [1,2]. Imported cases into Kenya following recent outbreaks in Angola and the Democratic Republic of Congo (DRC) (2015–2016) [2] demonstrate the potential for continued YF spread in the region. Improved understanding of the drivers of YF virus (YFV) transmission are urgently required through risk assessment to inform cost-effective preventive strategies.

Of the recognized YFV transmission cycles (sylvatic, rural, and urban cycles), outbreaks in the eastern African region have been described as sylvatic [3]. This is inclusive of the last documented YF outbreak in Kenya (1992–95) that implicated sylvatic Aedes vectors including Aedes simpsoni s.l. [4]. However, YFV transmission trends appear to be changing with demographic and environmental changes. For instance, the 2015/16 outbreaks in Angola and DRC were urban [2] and whether the East Africa region may face same fate in the future remains uncertain. The abundance of Ae. simpsoni s.l. vectors in peri-domestic human environments and associated Stegomyia indices have raised concerns about potential urban/rural risk of YFV transmission in Kenya [5]. Aedes simpsoni s.l. thus, has the potential to serve as bridge vector, moving the YFV from the sylvatic/rural to the urban transmission cycle. Aedes simpsoni complex comprises at least three described species including; Ae. lilii, Ae. simpsoni s.s., and the YF vector Ae. bromeliae [6]. To date, the distribution and relative contributions of the sub-species and/or subpopulations including bionomic characteristics in relation to YFV transmission dynamics remain poorly studied.

Demographic and societal changes including unplanned urbanization as occurring in Africa [7], are among important drivers of vector-borne disease spread. The consequent changes in urban architecture, via effects on environment, human abundance, vector density, and biting behavior have the potential to alter the vectorial capacity of mosquitoes and diseases transmission risk. We here, report data on survival and host feeding patterns of Ae. simpsoni mosquitoes in two areas of contrasting ecosystems: peri-urban, coastal Rabai, and rural, Rift Valley Kerio Valley, in Kenya. These parameters provide opportunities for vector infection and efficient transmission and are the two most sensitive indicators of disease transmission potential (i.e. vectorial capacity) [8,9]. Because, human vector feeding must precede outbreaks, knowledge of vector host-feeding behavior is essential to our understanding of the inter-epidemic maintenance patterns of YF. Furthermore, we assessed how these bionomic attributes vary among subspecies of the Ae. simpsoni complex.

Materials and methods

Ethics statement

The study received approval from the Scientific Ethics Review Unit (SERU) of the Kenya Medical Research Institute (Protocol NO. SSC 2787). Additionally, consent was sought verbally from household heads to set up traps around their homesteads.

Study site

Adult female Aedes simpsoni senso lato (s.l.) (hereafter as Ae. simpsoni) mosquitoes that had been collected from Rabai (peri-urban environment; human density ~600/km2) and from rural/sylvatic Kerio Valley (KV) (human density ~ 45/km2) as part of an arbovirus surveillance project were used in this study. Rabai (Kilifi County) is located northeast and ~25 km from Mombasa City in coastal Kenya while Kerio Valley with a history of YF outbreak [4] in the Rift Valley (Fig 1). The mosquito breeding habitats differ with Kerio Valley providing rural woodland setting with numerous tree holes for mosquito breeding as opposed to Rabai, with the peri-urban setting providing typical water storage containers for mosquitoes to breed and also outdoors breeding in plant axils and water receptables. Kerio Valley is an arid to semi-arid ecology that is sparsely populated. The main economic activities of the inhabitants include farming of crops like maize and cotton, and livestock keeping especially goats and cattle. Rabai is one of the seven administrative sub-counties of Kilifi county. The main economic activities in the area include subsistence agriculture, casual labor, crafts and petty trading.

Fig 1

Map of Kenya showing mosquito sampling sites: Kerio Valley (Baringo), Rabai (Kilifi) and Kajulu (Kisumu).

The map was designed using ArcMap 10.2.2 with the ocean and lakes base layer derived from Natural Earth (http://www.naturalearthdata.com/, a free GIS data source). The sample points were collected using a GPS gadget (garmin etrex 20, https://buy.garmin.com/en-US/US/p/518046), and the county boundaries for Kenya derived from Africa Open data (https://africaopendata.org/dataset/kenya-counties-shapefile, license Creative Commons).

Mosquito survey

Adult host seeking female mosquitoes were surveyed using CO2-baited BG Sentinel traps at three trapping periods with trapping exercise beginning about three weeks after the area first received rainfall continuously for at least a week. This timing was critical to maximize assessment of vector abundance, considering the predominant tree hole and plant axils breeding ecology of Ae. simpsoni [10]. The trapping periods were August-September 2019 (mean daily temperature = 25.4°C; mean daily rainfall = 0.58mm; relative humidity = 75.9%) and February 2020 (mean daily temperature = 27.4°C; mean daily rainfall = 0.30mm; relative humidity = 80.3%) in Rabai and November 2019 (mean daily temperature = 19.9°C; mean daily rainfall = 3.76mm; relative humidity = 72.4%) in KV. This trap type suitably targets Stegomyia mosquitoes which are active during the day [11]. Traps were set from morning to evening (06:30–18:00) on the same day, for at least 8 consecutive days per trapping period in peridomestic areas around homesteads. Sampling was designed to cover a large spatial area within each site. Traps were placed outdoors in the vegetation around human habitations and moved every other day to a new locality at least 500 m away. After retrieval, the mosquitoes were immobilised using triethylamine, sorted and transported in liquid nitrogen from the field to the Emerging Infectious Diseases laboratory at ICIPE, Nairobi. The mosquitoes were later identified morphologically to species level using published taxonomic keys [12] before storing in -80°C freezers for later use.

Parity rate, daily survival and longevity estimation

Mosquitoes after retrieval from -80°C were allowed to thaw on ice (4°C) and then dissected for parity after observing the degree of dilation of the tracheolar skeins of the avarioles [13]. After dissection, the remaining portion of each mosquito was preserved and processed for DNA extraction and molecular speciation (described below). Daily survival rates were derived from estimated parity rates for each sampling period as described previously [14] based on the formula: P = M, where P is the daily survival rate, M the parity rate and n the gonotrophic cycle (the number of days between emergence of adult females and first oviposition). A value of 3 days was assumed for this species [9]. Finally, the longevity (days) was estimated using the formula: 1/-lnL where L is the estimated survival [15].

Analysis of blood meals

Blood-fed specimens were individually dissected by separating the abdomen containing engorged blood from the head/thorax. The head/thorax were preserved for each mosquito and subjected to DNA extraction and molecular speciation (described below). Genomic DNA was extracted from the abdomen using the ISOLATE II Genomic DNA Kit (Bioline, Meridian Bioscience, Germany) as per the manufacturer’s instructions. DNA was amplified targeting a 500 bp fragment of the 12S mitochondrial rRNA gene using the primers 12S3F [5’-GGGATTAGATACCCCACTATGC-3’] and 12S5R [5’-TGCTTACCATGTTACGACTT-3’] [16] and as described previously [9]. PCRs in a 10 μl reaction volume comprised 2 μl 2x MyTaq Mix (Bioline, Meridian Bioscience, Germany), 10 μM of each primer, 0.2 U of Mytaq DNA polymerase and 1 μl of the template DNA (~20 ng). Thermal cycling conditions were 95°C for 3 min followed by 40 cycles at 95°C for 20 s, 59°C for 30 s and 72°C for 30 s and 72°C for 7 min. Amplicons were resolved on 1.2% agarose gel electrophoresis against a 100bp DNA HyperLadder (Bioline, Meridian Bioscience, Tennessee, USA). The PCR products were purified using the SureClean Plus kit (Bioline, Meridian Bioscience) and outsourced to Microgen (Netherlands) for Sanger sequencing using the forward primer. DNA sequences were compared using the BLAST algorithm and the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Species level identification was determined when sequences exhibited ≥ 98% identity spanning at least 300 bp as described previously [9].

Species typing of mosquitoes

Genomic DNA was extracted from the head/thorax of each blood fed specimen and remaining portion of selected parous mosquitoes as described previously. PCR was performed to identify the sibling species of Ae. simpsoni targeting the nuclear internal transcribed spacer 2 (ITS2) region using the primers ITS2A (5’-TGTGAACTGCAGGACACAT-3’) and ITS2B (5’-TATGCTTAAATTCAGGGGGT-3’) [17]. ITS2 is fast evolving as a marker of choice widely used for species-level discrimination [18-20] with vast sequence representation in public databases (e.g. GenBank). PCRs were performed in 20μl reaction volumes including 3μl 5x HOT FIREPol Blend Master Mix Kit (Solis Biodyne, Tartu, Estonia), 0.5μl of 10 μM forward and reverse primers, of 5x Mytaq HS mix polymerase and 20 ng DNA template in a ProFlex PCR systems thermocycler (Applied Biosystems, Foster City, CA, USA). The thermal cycling conditions were 95°C for 15 min followed by 40 cycles at 95°C for 30 s, 60°C for 30 s and 72°C for 45 s and 72°C for 7 min. Amplicons were confirmed by gel electrophoresis as described previously. Similarly, the PCR products were purified using SureClean Plus kit and outsourced for Sanger sequencing, in both the forward and reverse direction.

Sequences were viewed and edited in Chromas, embedded in MEGA v.6 [21] prior to querying the GenBank using BLASTn (www.ncbi.nlm.nih.gov/blast). Multiple sequence alignments of the resulting contiguous sequences were performed using ClustalW in MEGA v.6 with default parameters. Maximum likelihood (ML) trees were constructed with nodal support for the different groupings evaluated through 1000 bootstrap replications utilizing the Jukes and Cantor as best-fit model of sequence evolution.

Statistical analyses

The parity rate calculated as the percentage of parous mosquitoes to the total number dissected was established for each trapping period and comparisons made by Pearson chi-squared tests. The 95% confidence intervals (CIs) for the parity rates were estimated using binom.confint function. The human blood index is expressed as the proportion of blood-feeding on humans of the total number of blood-fed mosquitoes examined and established for subspecies and tested for significant differences using Pearson chi-squared tests. All analyses were performed at P = 0.05 using R v. 4.0.4 software [22].

Results

Parity, daily survival and age estimates

Overall parity rate was 85% (458/539; 95% CI 81.7–87.7%) broken down as follows: Rabai: August-September 2019 = 82.6% (71/86; 95% CI 73.2–89.1%); February 2020 = 85.1% (189/222; 95% CI 79.9–89.2%); KV: November 2019 = 85.7% (198/231; 95% CI 80.6–89.6%). The parity rate did not differ between the sites or sampling periods (P>0.05; Table 1). Estimated daily survival rates for Ae. simpsoni were high across the sampling periods in Rabai translating to longevity ranging from 15.8–18.8 days (Table 1). Aedes simpsoni in Kerio Valley had a higher longevity compared to Rabai, though the difference was not significant (P>0.05; Table 1).

Table 1

Estimated parity, daily survival and age for Ae. simpsoni mosquitoes.

Site	Survey period	Parity (% (n))	Daily survival rate	Longevity (days)
Rabai	August-September 2019	82.6 (86)a	0.9387	15.8
	February 2020	85.1 (222)a	0.9483	18.8
Kerio Valley	November 2019	85.7 (231)a	0.9504	19.7

a, not significant

Genetic composition of parous Ae. simpsoni s.l.

A subset of parous specimens (n = 43; Rabai = 37; KV = 6) randomly selected were profiled for DNA sequencing and phylogenetic analysis of the ITS2 region. Our findings show that most of the samples clustered with reference sequences of Ae. bromeliae with a strong bootstrap support (73%) (Fig 2). One specimen from Rabai grouped together with Ae. lilii in the Genbank and Ae. simpsoni samples included from Kajulu, in Kisumu County, western Kenya (Fig 2).

Fig 2

Maximum-likelihood tree derived from selected parous Ae. simpsoni ITS2 sequences using a JC model (216–313 nt).

Bootstrap values are shown above relevant nodes. Sequence of Ae. aegypti indicated as outgroup. The scale-bar indicates the number of substitutions per site. Taxon abbreviations represent sampling sites with numbers corresponding to specific sequence samples: RAB, Rabai; KV, Kerio Valley; KSM, Kisumu. Sequences have been submitted to GenBank with accession numbers OK339480–OK339521 (Aedes bromeliae), OK339522-OK339529 (Aedes lilii).

Blood meal feeding patterns

A total of 27 blood fed Ae. simpsoni s.l. mosquitoes were analysed from Kerio Valley (n = 9) and Rabai (n = 18), of which blood meal sources were successfully identified from 24 specimens. These represented 9 specimens from Kerio Valley and 15 from Rabai. The data revealed a total of 10 different hosts belonging to fairly large mammals (humans, goat, cow, domestic cat), rodents (grass rat, squirrel, mastomys mouse, African giant pouched mouse, mongoose) and reptile (lizard) (Fig 3). The host range was more diverse in Rabai than Kerio Valley (Fig 3). In both areas, Ae. simpsoni had fed more on humans, then squirrel (Rabai) followed by a lower representation of other host types (Fig 3). The overall human-blood-index (HBI) was 0.33 (8/24).

Fig 3

Aedes simpsoni host blood meals.

Association of Ae. simpsoni subspecies and human blood feeding

Next, we examined the relationship between subspecies of Ae. simpsoni s.l. and their influence on human blood feeding. A total of 27 Ae. simpsoni sl blood-fed specimens were profiled for phylogenetic analysis of sequenced ITS-2 region, with most of samples obtained from Rabai (n = 18), then Kerio Valley (n = 9). ITS2 sequences of the three blood-fed samples with unsuccessful blood meal data were included. The Ae. simpsoni sl samples resolved into 3 clades, with well supported bootstrap values (Fig 4). One of these clustered with Aedes bromeliae (GenBank No: KF135509) and had most of the samples from Rabai (n = 11) and Kerio Valley (n = 6). Clade II contained samples exclusively from Rabai (n = 4), while clade III had samples solely from Kerio Valley (n = 3). Thus, our findings show overwhelming representation of Ae. bromeliae among the blood-fed samples. Each of the clades exhibited human feeding tendency with no variation in the estimated human blood index (HBI) between the clades (P>0.05); clade I which clustered with Ae. bromeliae: (4/18); clade II: 2/3; clade III: 2/3 (Fig 4).

Fig 4

Maximum-likelihood tree derived from Aedes simpsoni blood-fed ITS2 sequences using a JC model (255–313 nt).

Bootstrap values are shown above relevant nodes. Sequence of Ae. aegypti indicated as outgroup. The scale-bar indicates the number of substitutions per site. Taxon abbreviations represent sampling sites with numbers corresponding to specific sequence samples: RAB, Rabai; KV, Kerio Valley. Sequences have been submitted to GenBank with accession numbers OK339453–OK339472 (Aedes bromeliae), OK339473-OK339479 (Aedes simpsoni sl).

Discussion

Here, we provide contemporary estimates of survival and blood feeding patterns for the important arboviral vector Ae. simpsoni s.l. collected from two areas with distinct ecosystems. High parity rates and hence survival rates/longevity were evident for this mosquito across the three trapping periods. Aedes simpsoni s.l. from Kenya was previously reported to be susceptible to YFV [23], and because infectious mosquitoes reflect the age structure of adult female mosquitoes [24], our findings suggest a high vectorial capacity for pathogen transmission by this mosquito species despite moderate human feeding rates.

Ae. bromeliae, which is the only known YFV vector among Ae. simpsoni mosquitoes in East Africa [6,25], has equally been suggested as the only human biting sibling species occurring in Kenya [6]. Ae. bromeliae appears dominant in the study areas based on our parity data (randomly selected) and blood fed specimens. While our result agrees with data reported in the literature [6,25], this trend might have been affected by its domestic/peri-domestic habits where sampling was conducted. The high survival/longevity found in the current study may contribute to the vectorial capacity of Ae. bromeliae. Age is the most sensitive vectorial capacity (a measure of pathogen transmission potential) parameter [8,9]. Higher YFV transmission risk relating to this subspecies is therefore, suggested in Kenya, although validation of its competence is still an important knowledge gap. Ae. simpsoni mosquitoes commonly breed in tree holes/plant leaf axils [5]. Its adaptation to breeding in water holding containers around human habitations [5] may contribute to seasonal abundance to enhance human risk as a result of increased vector human contact.

Our sequencing and phylogenetic analyses document for the first time the presence of Ae. lilii in Rabai, Kenya, thereby confirming previous speculations on the existence of this subspecies in the area [26]. The presence of Ae. lilii in Kenya was further supported by data from Kajulu, Kisumu in western Kenya (Fig 2). The results suggest geographic differences in the distribution of the sibling species in Kenya, warranting further studies to investigate this hypothesis. The very low numbers and absence of blood feeding for Ae. lilii, suggest minimal importance of this non-human biter [6,25], in disease transmission to humans in the study areas. Notably, we uncover potentially undescribed species (clades II and III) within the complex based on phylogenetic resolutions which displayed human feeding tendencies (Fig 4). The diversity of species in this complex could be much higher than previously thought which would require additional confirmatory studies. These studies could incorporate additional markers including analysis at the genomic level. Additionally, studies on the distribution and detailed ecology that could facilitate assessment of arboviral disease risk associated with distinct subspecies are warranted. Furthermore, a PCR-based method to distinguish the vector Ae. bromeliae from the non-vector Ae. lilii based on the ITS region was recently described [25]. Application of this protocol to our samples gave mixed and inconclusive results because several specimens amplified with both specific primer sets targeting these species.

Blood meal data showed feeding on diverse hosts for Ae. simpsoni with a corresponding moderate human blood index, a trend largely driven by Ae. bromeliae. The overall engorged specimens analyzed was relatively small (n = 24) reflecting the difficulty to trap blood-fed cohorts. This could as well have been affected by our trapping approach possibly biased towards host-seeking females. However, we attempted the newly developed Aedes gravid traps to enhance gravid and engorged mosquito cohorts [27] but had no catches. Thus, we suggest that further studies should incorporate resting collections to enhance the number of freshly engorged mosquitoes [28]. While a large sample size could allow for better inference on the trophic habit of these species, nonetheless, our study provides useful baseline data regarding host utilization by this heterogenous species group inferring opportunistic feeding in these arboviral disease foci. Feeding on humans by Ae. bromeliae and the potential uncharacterised species, increases the risk of transmission to humans of diverse vector-borne pathogens (e.g., yellow fever, dengue, chikungunya, and Zika viruses) including zoonotic ones circulating in livestock or rodent hosts.

YF outbreak preparedness and the Eliminate Yellow Fever Epidemics (EYE) strategy largely centers on vaccination scale-up, a costly venture that must be guided by justified risk. This focus must expand to include mosquito-based surveillance of critical parameters that define vectorial capacity in specific ecological contexts [8,9]. Combining laboratory experiments and field ecology while incorporating genotype-based analysis of vector populations should generate integrated data that could be modeled to predict potential YFV spread and re-emergence risk.

We conclude that blood-fed and parous specimens of Ae. simpsoni s.l. mosquitoes in the study areas were mainly Ae. bromeliae (the primary YFV vector in East Africa), and it appears to be the most dominant species in this complex. High parity rates and hence survival rates/longevity were evident for this mosquito across the three trapping periods, suggesting a high vectorial capacity for pathogen transmission by this mosquito. We report the presence of Ae. lilii in Kenya with potentially yet-to-be described species in the complex (clades II and III, Fig 4) displaying human feeding tendencies. We also infer a wide host feeding range on rodents, reptile, domestic livestock besides humans especially for Ae. bromeliae, a feeding trend that could likely expose humans to various zoonotic pathogens. Overall, we highlight the importance of precision surveillance data of vector populations through genotype-based analyses for enhanced disease risk prediction and to guide cost-effective interventions (e.g. YF vaccinations).

16 Dec 2021

Dear Dr. Tchouassi,

Thank you very much for submitting your manuscript "Influence of vectoral capacity and genetic structure of Aedes simpsoni on arbovirus transmission risk in East Africa" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Dear Dr. Tchouassi & co-authors,

Many thanks for submitting your manuscript to PLOS NTD. Apologies for the delay in getting this manuscript reviewed. Your manuscript has now been evaluated by three reviewers. The Reviewers agree that the study is of interest and all three provide feedback to improve the manuscript. I would like to ask you to respond to the concerns raised by the Reviewers and revise the manuscript accordingly (major revisions requested). All three reviewers pointed out that the manuscript in its current form, cannot draw firm conclusions regarding vectorial capacity since e.g. the vector competence for YFV of the mosquito species in question is unknown. It would be wonderful if the authors could provide experimental data to assess vector competence. However, if such data cannot be provided in this revision, the title and text of the manuscript should be modify to reflect this.

Please note that some Reviewers have provided additional comments in a word file.

Many thanks for your hard work - I look forward to seeing a revised version of this manuscript.

all the best,

Felix

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***********************

Dear Dr. Tchouassi & co-authors,

Many thanks for submitting your manuscript to PLOS NTD. Apologies for the delay in getting this manuscript reviewed. Your manuscript has now been evaluated by three reviewers. The Reviewers agree that the study is of interest and all three provide feedback to improve the manuscript. I would like to ask you to respond to the concerns raised by the Reviewers and revise the manuscript accordingly (major revisions requested). All three reviewers pointed out that the manuscript in its current form, cannot draw firm conclusions regarding vectorial capacity since e.g. the vector competence for YFV of the mosquito species in question is unknown. It would be wonderful if the authors could provide experimental data to assess vector competence. However, if such data cannot be provided in this revision, the title and text of the manuscript should be modify to reflect this.

Many thanks for your hard work - I look forward to seeing a revised version of this manuscript.

all the best,

Felix

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Objectives are clearly articulated and methods used are appropriate. Sample size is small and imbalanced for certain analysis, and thus prevents some statistical comparisons. However, the data is still informative.

Reviewer #2: The methods are adequate and well described. Sample size and statistics are correct.

However, the study design does not completely test the hypothesis of vector capacity. vector capacity encompasses vector competence and mosquito behavior and the study does not contain a measure of vector competence. Furthermore, there is no mention of earlier evaluation of the Ae bromeliae vector competence.

Reviewer #3: Local climate and environmental/landscape features of studied sites that may promote Ae. simpsoni s.l occurrence and possibly influence mosquito bionomics should be described, such as temperature, rainfalls pattern, humidity, etc, apart from tree holes and plat axile. Since this study has to do with vectorial capacity, which in turns depends on mosquito blood habits and host availability, a general account of human and other domestic animals census data should be described too.

Mosquito survey

Adult host-seeking female mosquitoes were surveyed using CO2-baited BG Sentinel traps at three trapping periods after the rains (lines 124-125). Clarify what do these trapping periods consisted about and why samplings were conducted only after rains? No details have been provided to clarify the procedure used to assign the traps to different sites.

Lines 126-129, inform of daily variations of temperature, rainfalls, humidity are results actually, so they should be placed in a specific section of the results.

Line 130 - Traps were set during the day (06:30 – 18:00h). Mosquito sampling as performed from morning to evening (06:30 – 18:00).

Parity rate, daily survival and longevity estimation

It is not clear whether mosquitoes were dissected after preservation or before preservation at -80ºC.

Statistical analyses

Proper statistical analysis to determine the degree of variability of genetic structure between species should be conducted as well.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: The results are clearly and completely presented. However, as mentioned for the methods, the study would greatly benefit from a measure of vector competence (by oral feeding Ae bromeliae with YFV) to meet their objective and main message of identifying Ae bromeliae as a capable vector for YFV.

Reviewer #3: Parity, daily survival and age estimates

I will ask authors to refrain from making statistical analysis to determine the significance of difference between data from different locations when the sampling timings are not similar because it is illogical. For instance, in Rabai the data used to estimate parity rate are from August-September 2019 and February 2020, whereas in KV there was only data from November 2019. There is no point here doing any sort of comparison to determine difference between sites. Do only descriptive analysis that should be restricted to each site. Don’t do any site-to-site comparison because the data were obtained in different contexts, there is no room for spatial comparisons with these data.

Genetic composition of parous Ae. simpsoni s.l.

This study didn’t characterize mosquito genome, what you did was conducting molecular identification of Ae. simpsoni sibling species by analysing the ITS2 region gene sequences. So, for clarity consider changing the title so it can read

Species composition of Ae. simpsoni complex.

Line 215, specify a subset of what sample size? How were the sample subsets selected?

Line 218 “…suggesting predominance of this species in both areas”. This is a discussion, not a result. Place it somewhere in the discussion.

Blood meal feeding patterns

Line 231 – indicate the total blood-fed mosquitoes from KV and Rabai that together made up 27 blood-fed specimens.

Discussion

The authors discuss the implications of their findings regarding arbovirus transmission risk at study sites, arguably due to high abundance of Ae. bromeliae, one of the most relevant YF vector in East African region. However, the authors have failed to emphasize while discussing result-by-result that in general the overall size of the mosquito sample was too small, that is, only 539 specimens were collected. This obviously precludes from making any strong conclusion regarding the variables analysed, such as, parity rate and longevity and source of blood meal. No strong inference can be also done on feeding habit when only 27 mosquitoes were analysed. The same applies to survival rate and longevity which also require observation of large number of dissected ovaries. Therefore, the results should be described and discussed very cautiously.

Lines 290 – 291 “The very low numbers and absence of blood feeding for Ae. lilii found in the current study confirms previous reports for the prevalence of this subspecies in eastern Africa”. There is no clear connection between this paragraph and the one before it. Consider rewriting or deleting.

Lines 294- 296, the authors implies that more sibling species may exist in the Ae. simpsoni group apart from those widely known three species. Please elaborate?

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: The study aims to provide information about the vectors for YFV in east africa. The authors collected mosquitoes from 3 geographically distinct areas in Kenya. The mosquito species was identified, their parity rates and the origin of the blood (human or other animals). These information incriminate Ae bromiliae from the Ae. simpsoni complex as a vector for arboviruses in these areas. However, there is a lack of information about the vector competence for Ae. bromaliae for the major arboviruses. From a quick literature search that was not available in the introduction, I found that Ae bromeliae vector competence is partially known although the available studies date back some time ago.

The current study would be more informative about the potential role of Ae bromeliae if they included vector comptence data. Importantly, the authors cannot state that they studied the influence of vector capacity of Ae simpsoni without including a measure of vector competence for YFV.

Reviewer #3: Conclusion

This section reflects the data reported

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: There is a mispelling in the title: "influence of vectorIal capacity".

l. 63-65: I do not understand this sentence.

l. 279-281: I also do not understand this sentence.

l. 235: there is a parenthesis missing.

l. 116: there is another parenthesis missing.

Reviewer #3: (No Response)

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: This study reports survey carried out in some villages in Kenya to understand the vectorial capacity and genetic structure of mosquito members of Aedes simpsoni group, a group comprising highly important, albeit neglected, YF vectors such as Ae. bromeliae. Although the data reported are relevant to broaden our undertesting of YF and associated arbovirus transmission ecological system, I think there are major aspect that must be addressed before this manuscript is considered for publication either in Plos Neglect or elsewehere.

In my understanding the manuscript title is highly misleading as the study hasn’t actually investigate the influence of vectorial capacity and genetic structure of Ae. simpsoni on arbovirus transmission. What author did was applying molecular analysis tools to investigate species composition of Ae. simpsoni complex. By reading the title I was expecting to find in the main manuscript description information about the amount and distribution of genetic variability between members of the Ae. simpsoni complex and possible link with their ability or not of transmitting arbovirus (YF) at study site. Additionally, the data reported doesn’t allow authors to make any inference about vectorial capacity as they only investigated mosquito blood-feeding habits and survival rate. Therefore, further information needs to be gathered so authors can really estimate vectorial capacity, these information include human-biting density (m), which cannot be inferred by a CO2-baited BG-traps, and human-biting habits (a). The product of these two parameters is the human-biting rate (ma). The human biting habit (a) is a product of human biting frequency (i.e., the length of the gonotrophical cycle) and the human blood index. As such, the manuscript doesn’t show convincingly that Ae. bromeliae has higher vectorial capacity although author have concluded that way. The inference of vectorial capacity has been done based solely on estimates of mosquito survival rate and human blood-feeding habits, lacking other fundamental parameters such as those aforementioned. Furthermore, no investigation of arbovirus infection was conducted on collected samples.

A such, in my perception, this study reports basically the occurrence, species composition, survival rate and blood-feeding habitats of Ae. simpsoni complex members. Therefore, result should be reported and discussed considering only the data produced and possible limitations for making generalization given the type of sampling approach applied and relatively lower number of specimens analysed (N= 539). For instance, only 27 blood-fed mosquitoes were analysed to determine the source of blood-meal. No serious conclusion can be made with such a smaller sample size.

Abstract

Abstract lines 34-36, “We here, address the need for improved understanding of vectorial capacity and genetic influence of Ae. simpsoni mosquitoes. Do the authors mean genetic influence of genetic structure?

Line 37- “Age structure was first estimated by parity in Ae. simpsoni”. This should be rewritten to read Age structure was determined based on the appearance of mosquito ovaries observed by dissection.

Lines 37-38 not clear for what purpose PCR and sequencing was used. What gene(s) or genetic markers were targeted?

Lines 39-41, Inference on vectorial capacity is not that simple, High human blood index doesn’t necessarily mean the species in question has consequently high vectorial capacity. Vectorial capacity is also a direct function of mosquito density relative to humans and human-biting habits (obtained by the product of frequency of mosquito biting and the human blood index also known as index of anthropophagy. This study doesn’t estimate any realistic vectorial capacity, it investigated blood-feeding habits and survival rates of Ae. simpsoni complex sibling species.

Introduction

This paper is about the influence of vectorial capacity and genetic structure of Aedes simpsoni on arbovirus transmission risk. However, I cannot see the rationale of studying the influence of vectorial capacity and genetic structure of Ae. simpsoni on transmission of arbovirus clearly presented in the introduction, particularly the extent to which arbovirus transmission can be influenced by vectorial capacity and genetic structure. The introduction emphasizes more aspects related to Ae. simpsoni bionomics, and virtually none account about the role of genetic structure on arbovirus transmission has been menioned. Moreover, it is not clearly stated what aspects of genetic structure authors have investigated. In fact, the influence of genetic structure hasn’t been mentioned has one of this study goals. It is clear to me that the goal was to study Ae. simpsoni survival rate and blood-feeding pattern, as it has been stated in lines 92-93.

Aedes simpsoni has been referred interchangeably as Ae. simpsoni s.l. and Ae. simpsoni. Ae. simpsoni has been sometimes referred to as Ae. simpsoni s.s which makes the reading difficulty to follow. Therefore, effort should be done to write the species name clearly so readers can understand the authors points.

Lines 77 – 78, “…last Kenyan YF outbreak”. I would suggest replacing the words “last Kenyan YF outbreaks” by one of the major YFV outbreak observed in Kenya so far (1992 - 95), as it looks to me that those were the final YF outbreak in Kenya, no outbreak will happen again in the future. This kind of contradicts what has been written in a previous paragraph (lines 72-73), concerning importation of YF cases from RDC and Angola into Kenya, in 2015-2016.

Line 80- “and this leaves the fate of East Africa unknown”. Suggest replacing by whether the East Africa region may face same fate in the future remains uncertain

Line 85- I think the correct spelling is Ae. lilii

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Submitted filename: Review.docx

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5 Jan 2022

Submitted filename: Review comments Jan522.docx

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13 Jan 2022

Dear Dr. Tchouassi,

Thank you very much for submitting your manuscript "Survival rate, blood feeding habits and sibling species composition of Aedes simpsoni complex: implications for arbovirus transmission risk in East Africa" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Dear authors,

Many thanks for revising the manuscript and answering the Reviewer's questions. I have some very minor comments that need to be addressed before acceptance, please find them below:

PNTD-D-21-01450_R1

Feedback on revised MS

Abstract:

lines 42-43 “and consequently, high vectorial capacity for pathogen transmission”: please rephrase ‘consequently’ to ‘potentially’ or similar. As the Reviewers indicated, caution should be taken in this study with regards to conclusions related to vectorial capacity (especially in the abstract). The word ‘consequently’ should therefore be revised. Same for line 72 in Author Summary.

MM

line 144, missing space between with and trapping.

Results:

line 279, parenthesis missing after mongoose.

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PLOS Neglected Tropical Diseases

Samuel Scarpino

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Dear authors,

Many thanks for revising the manuscript and answering the Reviewer's questions. I have some very minor comments that need to be addressed before acceptance, please find them below:

PNTD-D-21-01450_R1

Feedback on revised MS

Abstract:

lines 42-43 “and consequently, high vectorial capacity for pathogen transmission”: please rephrase ‘consequently’ to ‘potentially’ or similar. As the Reviewers indicated, caution should be taken in this study with regards to conclusions related to vectorial capacity (especially in the abstract). The word ‘consequently’ should therefore be revised. Same for line 72 in Author Summary.

MM

line 144, missing space between with and trapping.

Results:

line 279, parenthesis missing after mongoose.

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

13 Jan 2022

Submitted filename: Response to reviewer comments Jan1322.docx

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14 Jan 2022

Dear Dr. Tchouassi,

We are pleased to inform you that your manuscript 'Survival rate, blood feeding habits and sibling species composition of Aedes simpsoni complex: implications for arbovirus transmission risk in East Africa' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Felix Hol

Associate Editor

PLOS Neglected Tropical Diseases

Samuel Scarpino

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

Many thanks for taking care of these additional edits. Congrats on a nice manuscript - we're happy to publish it.

Felix

18 Jan 2022

Dear Dr. Tchouassi,

We are delighted to inform you that your manuscript, "Survival rate, blood feeding habits and sibling species composition of Aedes simpsoni complex: implications for arbovirus transmission risk in East Africa," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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