Article

# Aedes albopictus is not an arbovirus aficionado when feeding on cynomolgus macaques or squirrel monkeys

<figure><img src="image_1.png" /></figure>

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

Hélène Cecilia,
Benjamin M.
Althouse, Sasha R.
Azar, ..., Shannan L.
Rossi, Nikos
Vasilakis, Kathryn
A. Hanley

helene.cecilia3@gmail.com
(H.C.)
khanley@nmsu.edu (K.A.H.)

Highlights

Mosquitoes inoculated with dengue or Zika virus engorged similarly to controls

Uninfected mosquitoes engorged similarly on dengue/Zika-infected and control monkeys

A higher proportion of mosquitoes engorged on macaques than squirrel monkeys

Higher body temperature in squirrel monkeys induced higher engorgement rates
Article

# Aedes albopictus is not an arbovirus aficionado when feeding on cynomolgus macaques or squirrel monkeys

Hélène Cecilia,<sup>1,8,*</sup> Benjamin M. Althouse,<sup>1,2</sup> Sasha R. Azar,<sup>3,4</sup> Brett A. Moehn,<sup>1</sup> Ruimei Yun,<sup>3</sup>
Shannan L. Rossi,<sup>3,5,6,7</sup> Nikos Vasilakis,<sup>3,5,6</sup> and Kathryn A. Hanley<sup>1,*</sup>

SUMMARY

Viruses transmitted by *Aedes* mosquitoes (e.g., dengue [DENV], Zika [ZIKV]) have demonstrated high potential to spill over from their ancestral, sylvatic cycles in non-human primates to establish transmission in humans. Epidemiological models require accurate knowledge of the contact structure between hosts and vectors, which is highly sensitive to any impacts of virus infection in mosquitoes or hosts on mosquito feeding behavior. Current evidence for whether these viruses affect vector behavior is mixed. Here we leveraged a study on sylvatic DENV-2 and ZIKV transmission between two species of monkey and *Aedes albopictus* to determine whether virus infection of either host or vector alters vector feeding behavior. Engorgement rates varied from 0% to 100%, but this was not driven by vector nor host infection, but rather by the individual host, host species, and host body temperature. This study highlights the importance of incorporating individual-level heterogeneity of vector biting in arbovirus transmission models.

INTRODUCTION

Transmission dynamics of vector-borne pathogens are shaped by the contact structure between their arthropod vectors and their vertebrate hosts, which in turn depends upon vector attraction to individual hosts.<fnref n="1" /> The most important vector-borne pathogens in the context of global human health are those transmitted by mosquitoes, such as *Plasmodium* spp., dengue (DENV), and Zika (ZIKV) virus.<fnref n="2" /> Mosquitoes initially detect hosts at a distance through sensory cues such as carbon dioxide (CO₂).<fnref n="3" />⁻<fnref n="5" /> Once stimulated by CO₂, mosquitoes will target visual cues and odor plumes to find hosts.<fnref n="6" />,<fnref n="7" /> Ultimately, mosquitoes will make feeding decisions based on more proximal host cues, such as body heat, humidity, and skin odor, which is a product of volatile chemicals released by the skin microbiome.<fnref n="4" />,<fnref n="6" />,<fnref n="8" />

An effect of infection of host or vector on the feeding behavior of mosquitoes would have significant ramifications for patterns of pathogen transmission. To date, most studies testing the impact of infection on mosquito feeding have used *Plasmodium* spp., the parasite that causes malaria.⁹ As reviewed by Sanford and Shutler¹⁰ and Busula et al.,¹¹ most studies found that host infection with *Plasmodium* enhanced their attractiveness to mosquitoes. Switching perspective to the vector, laboratory studies have shown that *Plasmodium* infection changes mosquito feeding behavior, but such changes depend on the developmental stage of the parasite and *Plasmodium* species tested, among other factors.¹⁰,¹² For both the host and the vector, it remains unclear whether or how these laboratory findings translate to the field, and whether these observations reveal active manipulation by *Plasmodium* to enhance parasite fitness or simply a non-adaptive by-product of infection.¹⁰⁻¹²

In contrast to the rich body of work on *Plasmodium* spp., relatively few studies have investigated the effect of infection with arthropod-borne viruses (arboviruses) on mosquito feeding behavior. A review by Cozzarolo et al.¹³ showed mixed evidence for an impact of host infection on mosquito feeding. On one hand, several species of *Culex* mosquitoes did not differ in their attraction to house sparrows infected with either St. Louis encephalitis virus or Western equine encephalitis virus relative to uninfected house sparrows, and *Aedes taeniorhynchus* were not more attracted to sheep infected with Rift Valley fever virus (RVFV) than they were to uninfected sheep. On the other hand, *Culex annulirostris* were more attracted to chickens infected with Sindbis virus than they were to uninfected chickens, and *Culex pipiens* were more attracted to sheep infected with RVFV. Recently, Zhang et al.¹⁴ reported that mice and humans infected with DENV-2 or ZIKV produced

<footnote n="1">¹Department of Biology, New Mexico State University, Las Cruces, NM 88003, USA</footnote>

<footnote n="2">²Information School, University of Washington, Seattle, WA 98105, USA</footnote>

<footnote n="3">³Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USA</footnote>

<footnote n="4">⁴Center for Tissue Engineering, Department of Surgery, Houston Methodist Research Institute, Houston Methodist Hospital, Houston, TX 77030, USA</footnote>

<footnote n="5">⁵Center for Vector-Borne and Zoonotic Diseases, University of Texas Medical Branch, Galveston, TX 77555, USA</footnote>

<footnote n="6">⁶Institute for Human Infection and Immunity, University of Texas Medical Branch, Galveston, TX 77555, USA</footnote>

<footnote n="7">⁷Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA</footnote>

<footnote n="8">⁸Lead contact</footnote>

<footnote>*Correspondence: helene.cecilia3@gmail.com (H.C.), khanley@nmsu.edu (K.A.H.)</footnote>

<footnote>https://doi.org/10.1016/j.isci.2024.111198</footnote>
host cues that were more attractive for *Aedes aegypti* and *Aedes albopictus*, the major vectors of both viruses, than their uninfected counterparts.

Similar to studies of host infection, current evidence is inconclusive as to whether arbovirus infection of mosquitoes impacts their feeding behavior, as reviewed by Maire et al.¹⁵ Several studies have investigated the impact of DENV infection of Ae. *aegypti* on their tendency to feed on mice,¹⁶⁻²⁰ or guinea pigs.²¹ DENV-infected mosquitoes probed for longer periods of time than control mosquitoes in some studies,¹⁶,²⁰ but not others,¹⁷,²¹ showed a longer total feeding time (time to probe and engorge) in some studies,¹⁶,¹⁸ but not others,²¹ and were less likely to take a blood meal in some studies¹⁹ but not others.²⁰ To our knowledge, only one study²⁰ has tested the impact of DENV infection on probing efficiency; it showed DENV-infected Ae. *aegypti* probed more often than uninfected mosquitoes to achieve blood satiety. Studies on ZIKV so far have only focused on the impact it might have on Ae. *aegypti* host-seeking behavior, revealing significant but mixed (both up- and downregulated) effects on locomotor activity.¹⁵

A major limitation to most of the existing DENV and ZIKV studies is their reliance on rodents, which are not natural hosts for either virus. DENV and ZIKV originated in sylvatic cycles in zoonotic reservoir hosts, including non-human primates (NHPs)²²,²³ and arboreal *Aedes* mosquitoes in Asia and Africa, respectively.²²,²⁴ Both spilled over into humans and established human-endemic cycles transmitted by Ae. *aegypti* and Ae. *albopictus*.²⁵ Ae. *albopictus* may also be involved in spillover and spillback of both viruses.²⁶⁻²⁸

Here, we leveraged a study of sylvatic DENV-2 and ZIKV replication dynamics in both paleotropical (cynomolgus macaques, *Macaca fascicularis*) and neotropical (squirrel monkeys, *Saimiri boliviensis*) NHP hosts to test the effect of vector inoculation and host infection with each virus on vector feeding. Cynomolgus macaques are known to become naturally infected with both viruses,²³,²⁹ whereas neither virus has yet been detected in squirrel monkeys. We also took advantage of the experimental design to determine whether the immune response of NHPs, specifically cytokine concentrations, was notably affected by the exposure to bites of uninfected mosquitoes. While we do not expect cytokines to act on mosquitoes' decision to engorge, this complements our study as it is important to elucidate the immune response of hosts to mosquito bites *per se*.

## RESULTS

### Overview of the experimental design

The experiments that generated the data analyzed here have been described in detail by Hanley et al. 2024.³⁰ Figure 1 presents the experimental design and a detailed sampling scheme. Briefly, on day 0 of the experiment, cynomolgus macaques (*Macaca fascicularis*) or squirrel monkeys (*Saimiri boliviensis boliviensis*) were infected by being exposed to cartons containing the designated number of Ae. *albopictus*: macaques were exposed to cartons containing either 1 (low exposure) or 10 (high exposure) DENV-2-inoculated mosquitoes or 15 ZIKV-inoculated mosquitoes or 10 uninoculated mosquitoes (control). Squirrel monkeys were exposed to cartons containing either 15 DENV-2-inoculated mosquitoes or 15 ZIKV-inoculated mosquitoes or 15 uninoculated mosquitoes (control). Inoculation was performed intrathoracically 10 days prior to monkey exposure. Then, to assess transmission from infected hosts to mosquitoes, for both species, cartons of approximately 10 (for DENV-2-infected and control macaques) or 15 (all other treatments, note that number of mosquitoes per carton sometimes slightly exceeded 15) uninfected Ae. *albopictus* that had been starved of sucrose for 24 h were allowed to feed on NHP ears on the days designated in Figure 1. For both host species, blood was drawn at day -7 and at designated intervals from day 1-28 after the initial exposure to mosquitoes to quantify infectious viremia and cytokine concentrations. Although mosquitoes were starved and given no choice of hosts, engorgement rates nevertheless ranged from 0% to 100%, including on viremic NHPs (Figure S1).

### Impact of mosquito inoculation status on engorgement rates

In general, mosquito inoculation status had no significant effect on engorgement rate (Figure 2; Table S4), save that DENV-2-inoculated Ae. *albopictus* were less likely to engorge than control Ae. *albopictus* when feeding on squirrel monkeys (odds ratio [OR] = 0.31 [0.13, 0.71], *p* = 2.0e-4). We also detected an interaction between host species and mosquito inoculation status on feeding behavior, in that we found significantly higher engorgement of DENV-2-inoculated mosquitoes on cynomolgus macaques than on squirrel monkeys (OR = 6.46 [1.65, 25.3], *p* = 3.2e-4) but no difference between host species for ZIKV-inoculated or control mosquitoes (Table S4). For control mosquitoes, however, the general trend was in the same direction, with a higher proportion of mosquitoes engorging on cynomolgus macaques than squirrel monkeys. The selected model was a logistic regression model with a betabinomial error distribution, without random effects (Tables S5 and S6).

### Impact of host infection status and host species on engorgement rate

In a simple model comparing the experimental groups (combination of NHP species and infection status, and virus species), NHP infection status had no generally significant effect on engorgement rate (Figures 3A and 3B; Table S7), save that Ae. *albopictus* were less likely to engorge on ZIKV-infected cynomolgus macaques than on control cynomolgus macaques (OR = 0.45 [0.24, 0.86], *p* = 0.0011). When restricting the dataset to the range of days where viremia was detected for each virus (days 1–14 for DENV-2, 1–8 for ZIKV), the results remained qualitatively unchanged (Figure 3C). The selected simple model used a betabinomial error distribution and included a random intercept per monkey and day (Tables S8 and S9).

Furthermore, we found significantly higher Ae. *albopictus* engorgement rates on cynomolgus macaques than squirrel monkeys when considering control NHPs (OR = 2.07 [1.06, 4.05], *p* = 0.0043) or DENV-2-infected NHPs (OR = 2.77 [1.76, 4.38], *p* = 4.2e-9), but not ZIKV-infected NHPs (*p* = 0.55).
<figure><img src="image_4.png" /></figure>

A

Day 0

<figure><img src="image_5.png" /></figure>

Effect of vector
inoculation status

<figure><img src="image_6.png" /></figure>

B

Cynomolgus
macaques

<figure><img src="image_7.png" /></figure>

Control

DENV low exposure

DENV high exposure

ZIKV

N = 2 ♀ 2 ♂

N = 2 ♀ 2 ♂

N = 3 ♀ 2 ♂

N = 2 ♀ 1 ♂

<figure><img src="image_8.png" /><figcaption>**Figure 1.** Experimental infections of cynomolgus macaques and squirrel monkeys with sylvatic dengue or Zika virus</figcaption></figure>

In the overview (A), black represents uninfected vectors/hosts and red represents inoculated vectors or infected hosts. In the detailed sampling scheme (B), letters denote: C, serum sampled for cytokine quantification; M, mosquito feeding; V, serum sampled for viremia quantification; W, weight. A and B indicate cohorts, in the case of squirrel monkeys. The monkey and mosquito images are licensed from Shutterstock. †: only measured in control and DENV-2 infected cynomolgus macaques. ‡: only measured for ZIKV-infected cynomolgus macaques. °: only measured for cohort B.

Impact of host infection and biology as well as time of day on engorgement rate

The complete model tested the effect of host species, sex, weight, body temperature at the time of feeding, virus, and viremia, as well as time of day, on engorgement rates. Two-way interactions between host species and virus, host species and weight, and host species and body temperature were included. The model highlighted a significant, positive effect of host body temperature on Ae. albopictus engorgement on squirrel monkeys (p = 0.0061), but not on cynomolgus macaques (p = 0.59). The relationship between host body temperature and engorgement rates is showed in Figure 4. No other variables had a significant effect (Table S10). The selected complete model used a betabinomial error distribution and included a random intercept per monkey and day (Tables S11 and S12).

Degree of inter-individual variation in mosquito engorgement rates among hosts in this study compared to approach rates
in free-living *Aedes albopictus*

In our experiments, engorgement rates varied substantially within and between NHPs (Figures 5 and S1). The coefficient of variation per NHP ranged from 0.22 to 0.88, with a mean of 0.45. In a study measuring the approach of free-living mosquitoes to humans,³¹ the number
<figure><img src="image_9.png" /><figcaption>**Figure 2. Engorgement rates of *Aedes albopictus* on day 0 of the experiments, depending on the mosquito inoculation status and NHP species**</figcaption></figure>

(A) Probabilities of feeding predicted by the model per group (point), along with 95% confidence intervals, assuming a duration of mosquito exposure of 6 min (median of all exposures), with results of statistical comparisons indicated above brackets, with ns: *p* > 0.05, **: 0.0001 < *p* < 0.001, results of *post hoc* Wald tests (or z-test, details in Table S4). From left to right, *n* (number of NHP) = 3, 5, 3, 15, 4, 7.

(B) Raw data (one point = one NHP) colored by mosquito inoculation status (gray triangle: control, green circle: DENV-2, blue square: ZIKV), with one boxplot per group. Box boundaries indicate the 25th (bottom) and 75th (top) percentiles. The line within the box marks the median. Whiskers above and below the box indicate the 10th and 90th percentiles, respectively. Duration of mosquito exposure varies but is not shown here. The monkey images are licensed from Shutterstock. See also Tables S4–S6.

of approaching female *Ae. albopictus* ranged from 0 to 6 (Figure S3) and the coefficient of variation ranged from 0 to 2, with a mean of 1.43.

**Repeated exposures to bites of uninfected mosquitoes shaped EGF and MIF concentrations in cynomolgus macaques**

We considered the effects of mosquito bites on cytokine concentrations in control NHPs, both in the short term (the number of bites received the day prior for cynomolgus macaques, and 2 days prior for squirrel monkeys, due to alternating days of exposure) and the long term (the cumulative number of bites received in the last seven days for both species). We included all cytokine measures from day −7 to day 28, and the short-term and long-term exposure variables varied concurrently (Figure S4), accounting for days with no mosquito bites.

In control cynomolgus macaques, daily epidermal growth factor (EGF) and macrophage migration inhibitory factor (MIF) concentrations were significantly associated with short-term and long-term exposure to uninfected mosquito bites. For EGF, a generalized additive mixed effect model was selected, estimating linear effects of both short-term and long-term bite exposure, and splines to fit individual intercepts. For MIF a generalized additive mixed effect model was selected, estimating a linear effect of long-term bite exposure, and splines for short-term bite exposure and individual intercepts. For EGF, the concentrations increased by 0.02 [0.01, 0.03] log₁₀ pg/µL per additional bite the day prior (*p* = 2.15e−4, Figure 6A; Table S13). For MIF, the relationship with short-term exposure was non-linearly positive, with a saturation after about 6 bites the day prior (*p* = 0.005, Figure 6B; Table S13). Both cytokines decreased linearly with long-term bite exposure (−0.006 [−0.008, −0.004] log₁₀ pg/µL per additional bite in the last seven days for EGF, *p* = 8e−6, −0.010 [−0.014, −0.005] for MIF, *p* = 2.3e−4, Figures 6C and 6D; Table S13). In both cases, the models captured variation in the data satisfactorily (Figures S5 and S6). All measures were above the limit of detection for these cytokines.
<figure><img src="image_10.png" /><figcaption>**Figure 3. Engorgement rates of uninfected *Aedes albopictus* depending on NHP infection status and species**</figcaption></figure>

(A) Probabilities of feeding predicted by the simple model per group (point), along with 95% confidence intervals, assuming a duration of mosquito exposure of 6 min (median of all exposures), with results of statistical comparisons indicated above brackets, with ns: *p* > 0.05, **: 0.001 < *p* < 0.01, ***: 0.0001 < *p* < 0.001, results of post hoc Wald tests (or z-test, details in Table S7). From left to right, *n* (number of batches of mosquitoes) = 36, 81, 33, 26, 60, 57.

(B) Raw data (one point = a single batch of mosquitoes fed on a given NHP on a given day) colored by NHP infection status (gray triangle: control, green circle: DENV-2, blue square: ZIKV), with one boxplot per group. Duration of mosquito exposure varies but is not shown here. Data from day 28 excluded (see STAR Methods).

(C) Raw data, same as (B) except that for infected groups, we restrict the data to the range of days where viremia was detected for each virus (days 1–14 for DENV-2, 1–8 for ZIKV). In (B) and (C), box boundaries indicate the 25th (bottom) and 75th (top) percentiles. The line within the box marks the median. Whiskers above and below the box indicate the 10th and 90th percentiles, respectively. The monkey images are licensed from Shutterstock. See also Tables S7–S9.

Daily tumor growth factor β (TGF-β) and monocyte chemoattractant protein-1 (MCP-1, also called CCL2) concentrations were also significantly, negatively associated with long-term bite exposure (Figures S7–S9; Table S13). For both TGF-β and MCP-1, a generalized additive mixed effect model was selected, estimating linear effects of both short-term and long-term bite exposure, and splines to fit individual intercepts. For TGF-β, the concentration decreased by 0.0036 [−0.0060, −0.0013] log₁₀ pg/µL per additional bite received in the last 7 days (*p* = 0.0054, Figure S7A), and for MCP-1, it decreased by 0.0039 [−0.0064, −0.0013] log₁₀ pg/µL per additional bite received in the last 7 days (*p* = 0.0058, Figure S7B). In both cases, the models captured variation in the data satisfactorily (Figures S8 and S9). All measures were above the limit of detection for these cytokines.

In cynomolgus macaques, interleukins 1β, 4, 5, 10, 17, and VEGF could not be tested because all measures were below the limit of detection. All other tested cytokines showed no significant association with bite exposure. In squirrel monkeys, we could only test I-TAC, MIF, MIP-1β, and RANTES, as all other cytokines had too many observations below the limit of detection. These 4 cytokines showed no significant association with bite exposure. All results are presented in Table S13.
<figure><img src="image_11.png" /><figcaption>**Figure 4.** Relationship between host body temperature and *Aedes albopictus* engorgement rates, in cynomolgus macaques (left column) and squirrel monkeys (right column)</figcaption></figure>

Raw data (one point = one NHP on one day) colored by NHP infection status (gray triangle: control, green circle: DENV-2, blue square: ZIKV). Note that the same set of control animals are shown for same species experiments (A, C for cynomolgus macaques, B, D for squirrel monkeys). The duration of mosquito exposure varies but is not shown here. Data from day 28 excluded (see STAR Methods). The monkey images are licensed from Shutterstock. See also Figure S2 and Tables S10–S12.

DISCUSSION

Identifying the factors that shape the feeding behavior of arbovirus vectors is critical for advancing model-based predictions of arbovirus spill-
over, spillback, and between-host transmission. Here, we studied the engorgement rates of Ae. *albopictus*, a likely bridge vector between
sylvatic and urban cycles of arbovirus transmission, on two NHP species, investigating the effect of vector inoculation and host infection
with sylvatic strains of DENV-2 or ZIKV.

We did not find any systematic effect of vector inoculation with DENV-2 or ZIKV on the feeding behavior of Ae. albopictus, save for a ten-
dency of DENV-2-inoculated Ae. albopictus to engorge at lower frequency on squirrel monkeys than control Ae. albopictus. Wei Xiang et al.²⁰
showed that DENV-2-infected Ae. aegypti were more attracted to uninfected mice than control mosquitoes but were less successful when
probing, necessitating more probes to feed to repletion. In combination, these two effects resulted in similar blood-feeding rates between
infected and control mosquitoes, over an observation period of 30 min. Because mosquitoes in our experiments were only given a limited
time to engorge (due to the need to minimize time under anesthesia for the NHPs), impaired probing could lead to lower rates of engorge-
ment, even if inoculated mosquitoes were more attracted to hosts. We note that we were not able to observe probing in our experiments.
Additionally, in the Wei Xiang et al. study, mosquitoes were infected by feeding on a blood meal containing virus, recapitulating all steps of
mosquito infection, whereas in our study, mosquitoes were inoculated intrathoracically with virus, a procedure that bypasses the mosquito
midgut. Furthermore, in our experiments, control mosquitoes were not subjected to intrathoracic injection, which could have confounded
effects of virus inoculation on feeding. However, intrathoracic inoculation was conducted 10 days prior to feeding on NHPs, giving mosqui-
toes substantial time to recover from the procedure.

We also did not find any universal effect of host infection with sylvatic DENV-2 or ZIKV on Ae. albopictus engorgement rates, with the exception that mosquitoes engorged significantly less frequently on ZIKV-infected macaques than control macaques. These results contrast with those of a recent study by Zhang et al.,¹⁴ who showed that, in a dual-choice olfactometer assay, Ae. aegypti and Ae. albopictus were more attracted to host cues produced by DENV-2- or ZIKV-infected mice or humans relative to controls, albeit mosquitoes were not allowed to feed
<figure><img src="image_12.png" /><figcaption>**Figure 5.** Engorgement rates of *Aedes albopictus* on individual NHPs, normalized by duration of exposure, depending on monkey infection status and species</figcaption></figure>

One boxplot per NHP, colored by NHP infection status (gray: control, green: DENV-2, blue: ZIKV), with raw data (triangle: control, circle: DENV-2, square: ZIKV). Box boundaries indicate the 25th (bottom) and 75th (top) percentiles. The line within the box marks the median. Whiskers above and below the box indicate the 10th and 90th percentiles, respectively. Red points indicate feedings on animals that were detectably viremic or that resulted in transmission to mosquitoes. Data from day 28 excluded (see STAR Methods). The monkey images are licensed from Shutterstock. See also Figures S1 and S3.

in this assay. These results cannot be compared to ours because of differences in protocols. However, in another experiment by Zhang et al., Ae. aegypti were allowed to directly access mice, and this experiment found increased feeding on infected hosts. Differences in the findings of Zhang et al. and the current study may reflect differences in the vector species used (Ae. aegypti in Zhang et al.,¹⁴ Ae. albopictus in the present study), the host species used (immunodeficient mice in Zhang et al.,¹⁴ natural NHP hosts in the present study), the ecotype of virus used (human-endemic in Zhang et al.,¹⁴ sylvatic in the present study), or host infection route (needle delivery in Zhang et al.,¹⁴ mosquito bite in the present study). Cozzarolo et al.¹³ suggested that the ideal method for interrogating mosquito feeding preferences would be dual-choice experiments where vectors are allowed to bite and hosts allowed to defend themselves. When protocols ethically require animals to be anesthetized, as was the case here and in Zhang et al., hosts cannot defend themselves. Nevertheless, in our experiments NHPs did not display any signs or behavior associated with illness,³⁰ which could have driven a possible preference toward infected animals. The use of anesthesia raises another caveat for our study, as Buchta et al.³² showed that anesthesia, by dropping core body temperature, impacted mosquito feeding, in experiments performed on guinea pigs with *Anopheles stephensi*. They recommended the use of a warming device to maintain normothermic body temperature of the host when conducting feeding experiments with mosquitoes. In our case, core body temperature was maintained in squirrel monkeys by placing them on a preheated warming blanket. This was not done for cynomolgus macaques, but mosquito exposure occurred shortly after the anesthesia so that their temperature had not yet dropped significantly (Figure S2). Importantly, the anesthesia procedure for control and infected monkeys was identical, so, while anesthesia could have affected overall feeding rate, it could not have biased comparisons between host infection status. It will be important to disentangle the possible long-range and short-range effects of host infection status on mosquito attraction and actual bites, possibly with a similar framework to Wei Xiang et al.,²⁰ to see how these effects impact the overall contact rate and opportunities for virus transmission.

A recurrent effect on mosquito engorgement in our study was host species, with more engorgement on cynomolgus macaques, which was evident in control NHPs and DENV-2 infected NHPs but not in ZIKV-infected NHPs. In day 0 mosquitoes, this species difference was only seen from DENV-2-inoculated mosquitoes, but this could be due to smaller sample sizes. In natural settings, Ae. albopictus is known to feed on a wide range of hosts,³³ and preferences can be driven by visual cues such as body size.³⁴ However, in our setup mosquitoes could not detect hosts from a distance, and weight (a proxy of body size) did not explain observed engorgement rates according to our model. To understand why mosquitoes are more eager to engorge on certain NHP species than others, other host-level factors such as host odor,⁸,⁹ which is in part shaped by skin microbiome,³⁵,³⁶ should be investigated. Studies so far have focused on characterizing similarities and differences in odor profiles between humans and NHPs,⁸,³⁵⁻³⁷ as well as between apes and monkeys,³⁵,³⁶ but comparisons between monkey species that could explain possible trophic preferences of mosquitoes in sylvatic environments have not been made. Whether host or vector infection can counter-balance a possible trophic preference of Ae. albopictus toward certain NHP species needs to be investigated further.

After accounting for other possible drivers of engorgement, the only variable that stood out was host body temperature, with a positive effect in squirrel monkeys only. Studies usually focus on mosquitoes' ability to detect warm-blooded hosts from a background temperature,³⁸,³⁹ rather than the effect of host body temperature per se. Cynomolgus macaques' body temperature was on average 1.2 °C colder that of squirrel monkeys at the time of mosquito feeding, but cynomolgus macaques were housed in rooms on average 2 °C colder than squirrel monkeys. We unfortunately could not measure the exact difference between host body temperature and room temperature, and ideally
<figure><img src="image_13.png" /><figcaption>**Figure 6.** Analysis of the relationship between repeated exposures to bites of uninfected mosquitoes and cytokine concentrations</figcaption></figure>

In control cynomolgus macaques, concentrations of cytokines EGF (A and C) and MIF (B and D) are significantly associated with the number of uninfected mosquitoes which engorged the day before (A and B) and in the last 7 days (C and D). Results from generalized additive mixed effect models, estimating an intercept per NHP. Thick black lines and shading show the population trends with uncertainty. Colored lines are individual fits. Fits for a given exposure variable (short-term or long-term) are computed with the other variable fixed (0 for short-term, 10 for long-term), which is why data points are not plotted (short-term and long-term variables varied concomitantly in the experiments). Note that y axis scales differ between cytokines (A and C vs. B and D) and that x axis scales differ between exposure variables (A and B vs. C and D). The monkey images are licensed from Shutterstock. See also Figures S4–S9.

the difference in use of a heating blanket between species should be corrected. Higher body temperature has been suggested as a possible explanation for why pregnant women were more attractive to malaria vectors than non-pregnant women,⁴⁰ with an average difference of temperature between their abdomens of 0.7 °C. In the present study, squirrel monkeys' body temperature ranged from 37.82 °C to 40.28 °C at the time of mosquito feeding, and the associated predicted probabilities of engorging ranged from 0.53 to 0.74. A positive effect of body heat on mosquito engorgement might be linked to an increased release of volatile compounds.⁴⁰ Overall, our study depicts mosquito engorgement as a highly variable, multi-factorial phenomenon,⁴¹ even in controlled conditions.

To understand variation in disease transmission, focusing on a single event in the mosquito feeding process, namely engorgement, as we did, following a relatively long exposure to a host, might not be sufficient. This outcome has the advantage of being easy to measure, but it does not represent the feeding behavior in its entirety. Other metrics, such as the time to first bite, time between bites, and duration of probting and feeding, have been shown to play a role in pathogen transmission rate.¹⁵,²⁰,⁴² Several mathematical models have sought to finely represent the biting behavior of mosquitoes and its influence on transmission dynamics.⁴²⁻⁴⁵ Nonetheless, in epidemiological models of vector-borne diseases transmission dynamics, the biting rate is most often represented as a constant, influenced neither by abiotic (e.g., temperature) nor by biotic (e.g., host species, infection status, or allometry¹⁶,⁴⁷) factors. Another common modeling hypothesis is to assume that a single blood meal takes place per gonotrophic cycle, whereas imbibing multiple blood meals is a common behavior in several species, including Ae. aegypti.⁴⁸,⁴⁹ This has important implications, as recent studies have showed that successive feeding episodes could enhance viral dissemination within mosquitoes.⁵⁰⁻⁵² Vector preference, particularly in multi-host systems, can have important epidemiological consequences, which can be studied conceptually through mathematical models.⁵³,⁵⁴ More studies are needed, both in controlled experimental settings and in the field, to understand the underlying factors that influence mosquito biting habits.

The variability of Ae. albopictus engorgement per NHP and between NHPs was striking in our experiments, and the occasionally low engorgement rates observed were surprising to us as mosquitoes had no other choice of host. Engorgement is the culmination of successive mosquito decisions to approach, land, and probe. To contextualize our observed variation in engorgement, we compared it to the variation observed in the approach rate of free-living Ae. albopictus to different human collectors in the field. The latter was more extreme, which likely reflects the inherently greater variation of field compared to laboratory experiments as well as the greater number of cues and environmental factors that may influence approach compared to engorgement. For both processes, we highlight the importance of incorporating individual-level heterogeneity of vector's feeding behavior in models at the population scale, even in the absence of mechanistic drivers.

When they bite, mosquitoes deliver salivary gland proteins, a complex cocktail that can impact host immunity⁵⁵ and, if a virus is also delivered, viral replication dynamics.⁵⁶⁻⁵⁸ While the impact of mosquito saliva on host susceptibility to arboviruses and subsequent arbovirus
<figure><img src="image_14.png" /></figure>

dynamics has been well studied,⁵⁹ particularly in mice, the cytokine response of NHPs to the bite of uninfected mosquitoes has received relatively little attention. We took advantage of data collected in the current study to begin to close this knowledge gap. In control cynomolgus macaques, among 29 cytokines analyzed, we identified short-term (e.g., bites occurred 1 day prior) positive associations between uninfected mosquito bites and EGF and MIF concentrations, as well as long-term (bites occurred over a seven day period prior) negative associations with EGF, MIF, TGF-β, and MCP-1 concentrations, while the remaining 25 cytokines assayed showed no significant responses to repeated mosquito bites. This is consistent with recruitment of leukocytes to the bite site (EGF, MCP-1) to resolve inflammation (MIF, TGF-β), which slows down over time as bites keep happening, but the response is already in place. In their study on humanized mice bitten by uninfected mosquitoes, Vogt et al.⁵⁵ noticed a decrease in MCP-1 concentrations 6 h post-bite and an increase 7 days post-bite. They observed no change in EGF 24 h post-bite and did not measure MIF and TGF-β. Here, we focused on the cytokine response in the absence of pathogen transmission, but, in studies of infected animals, IL-4 and IL-10 are often cited as being upregulated in response to arbovirus infection through mosquito bites.⁵⁶⁻⁵⁸,⁶⁰ In our study, control NHPs of both species produced concentrations of these two cytokines below the limit of detection over the whole duration of the experiment, suggesting that this upregulation does not happen in the absence of arbovirus transmission. Although cynomolgus macaques were housed indoors for several months before the start of the study, they initially came from Mauritius and therefore may have had previous exposure to Ae. albopictus. Future experiments could pursue skin biopsies to investigate the immediate mobilization of these cytokines following a mosquito bite.⁶¹

## Limitations of the study

Our study suggests that inoculation of *Aedes albopictus* with sylvatic DENV and ZIKV does not increase the tendency to engorge on the natural sylvatic hosts of these viruses. We did not test the infection status of unengorged mosquitoes, which necessitates that we limit our inference to inoculation rather than infection. Intrathoracic inoculation was used to standardize infection of mosquitoes and maximize our chances of infecting monkeys, but, to get closer to field conditions, mosquitoes should be infected through a natural blood meal. Our study also suggests that sylvatic hosts' infection with orthoflaviviruses does not lead to increased likelihood of Ae. albopictus engorgement. Our protocol is unique, in that it used sylvatic strains of virus coupled with natural hosts of DENV and ZIKV. Still, this research was conducted under laboratory conditions, which offer opportunities for control and inference of causality but inherently limit the number of cues and environmental factors that may influence mosquito feeding behavior. Furthermore, we focused on a single measure of feeding, the proportion of engorged mosquitoes observed after a period of exposure to an anesthetized host, which does not capture the entire arc of mosquito attraction, probing, and feeding. This all may limit the generalizability of our findings, as well as testing only one mosquito species. We did not test the monkeys for anti-mosquito antibodies when we acquired them, which could have added more nuance to our analysis of the effects of mosquito bites on cytokine levels. Further research could leverage more sophisticated tools to observe mosquito feeding behavior in its entirety (approach, land, probe), combined to an identification of individual-level factors (e.g., microbiome, odor profile) responsible for attractiveness to mosquitoes.

## RESOURCE AVAILABILITY

### Lead contact

Further information and requests for resources should be directed to the lead contact, Hélène Cecilia (helene.cecilia3@gmail.com).

### Materials availability

This study did not generate new unique reagents.

### Data and code availability

* Raw and analyzed data have been deposited at Zenodo and are publicly available as of the date of publication. DOIs are listed in the key resources table.

* All original code has been deposited at Zenodo and is publicly available as of the date of publication. DOIs are listed in the key resources table.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

This research was funded by grant 1R01AI145918-02 (to K.A.H., N.V., B.M.A., and S.L.R.) and in part by the Centers for Research in Emerging Infectious Diseases "The Coordinating Research on Emerging Arboviral Threats Encompassing the Neotropics (CREATE-NEO)" grant 1U01AI151807 from the US National Institutes of Health (to N.V. and K.A.H.). Squirrel monkeys acquired from the UT MD Anderson Cancer Center, Michael E. Keeling Center for Comparative Medicine and Research, were supported by the NIH grant 5P40OD010938-36 prior to sale to UTMB. Fellowship 709139 from CONACYT (Consejo Nacional de Ciencia y Tecnología, Government of Mexico) was used to produce data on mosquito approach in the field in Manaus, Brazil. We thank Eduardo Hernandez Acosta for his assistance in interpreting the data for the free-living mosquitoes.

## AUTHOR CONTRIBUTIONS

Conceptualization: H.C., B.M.A., N.V., and K.A.H. Methodology: H.C., B.M.A., and K.A.H. Investigation: S.R.A., B.A.M., R.Y., K.A.H., N.V., and S.L.R. Visualization: H.C. Formal analysis: H.C. Software: H.C. Funding acquisition: N.V. and K.A.H. Project administration: N.V., K.A.H., and S.L.R. Supervision: B.M.A. and K.A.H. Writing – original draft: H.C., B.M.A., and K.A.H. Writing – review and editing: H.C., B.M.A., S.R.A., B.A.M., R.Y., S.L.R., N.V., and K.A.H. All authors contributed to the article and approved the submitted version.
## DECLARATION OF INTERESTS

The authors declare no competing interests.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* Non-human primates (NHPs)

* Mosquitoes

* Cell lines and virus strains

* METHOD DETAILS

* Infection of mosquitoes and monkeys

* Monitoring viremia, cytokine responses, and transmission

* QUANTIFICATION AND STATISTICAL ANALYSIS

* Engorgement and mosquito viral inoculation

* Preliminary observations

* Engorgement and host infection

* Comparison with free-living *Aedes albopictus*

* Repeated mosquito bites and cytokine response

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.111198.

Received: March 12, 2024

Revised: June 27, 2024

Accepted: October 15, 2024

Published: October 19, 2024

## REFERENCES

1. Thongripong, P., Hyman, J.M., Kapan, D.D., and Bennett, S.N. (2021). Human-Mosquito Contact: A Missing Link in Our Understanding of Mosquito-Borne Disease Transmission Dynamics. Ann. Entomol. Soc. Am. 114, 397–414. https://doi.org/10.1093/ aesa/saab011.

2. World Health Organization (WHO) (2014). A global brief on vector-borne diseases (World Health Organization), p. 54.

3. Lorenz, L.M., Keane, A., Moore, J.D., Munk, K.J., Seeholzer, L., Mseka, A., Simfukwe, E., Ligamba, J., Turner, E.L., Biswaro, L.R., et al. (2013). Taxis assays measure directional movement of mosquitoes to olfactory cues. Parasit. Vectors 6, 131. https://doi.org/10. 1186/1756-3305-6-131.

4. Liu, M.Z., and Vosshall, L.B. (2019). General Visual and Contingent Thermal Cues Interact to Elicit Attraction in Female *Aedes aegypti* Mosquitoes. Curr. Biol. 29, 2250–2257.e4. https://doi.org/10.1016/j.cub.2019.06.001.

5. Sorrells, T.R., Pandey, A., Rosas-Villegas, A., and Vosshall, L.B. (2022). A persistent behavioral state enables sustained predation of humans by mosquitoes. Elife 11, e76663. https://doi.org/10.7554/eLife.76663.

6. Cardé, R.T. (2015). Multi-Cue Integration: How Female Mosquitoes Locate a Human Host. Curr. Biol. 25, R793–R810. https://doi. org/10.1016/j.cub.2015.07.057.

7. Vinauger, C., Van Breugel, F., Locke, L.T., Tobin, K.K.S., Dickinson, M.H., Fairhall, A.L., Akbari, O.S., and Riffell, J.A. (2019). Visual-Olfactory Integration in the Human Disease Vector Mosquito *Aedes aegypti*. Curr. Biol. 29, 2509–2516.e5. https://doi.org/10.1016/j. cub.2019.06.043.

8. Tchouassi, D.P., Jacob, J.W., Ogola, E.O., Sang, R., and Torto, B. (2019). *Aedes vector*-host olfactory interactions in sylvatic and

domestic dengue transmission environments. Proc. Biol. Sci. 286, 20192136. https://doi.org/10.1098/rspb.2019.2136.

9. Martinez, J., Showering, A., Oke, C., Jones, R.T., and Logan, J.G. (2021). Differential attraction in mosquito-human interactions and implications for disease control. Phil. Trans. R. Soc. B 376, 20190811. https://doi. org/10.1098/rstb.2019.0811.

10. Sanford, T., and Shutler, D. (2022). Manipulation by Plasmodium Parasites of Anopheles Mosquito Behavior and Human Odors. Acta Parasit 67, 1463–1470. https:// doi.org/10.1007/s11686-022-00621-6.

11. Busula, A.O., Verhulst, N.O., Bousema, T., Takken, W., and De Boer, J.G. (2017). Mechanisms of Plasmodium-Enhanced Attraction of Mosquito Vectors. Trends Parasitol. 33, 961–973. https://doi.org/10. 1016/j.pt.2017.08.010.

12. Stanczyk, N.M., Mescher, M.C., and De Moraes, C.M. (2017). Effects of malaria infection on mosquito olfaction and behavior: extrapolating data to the field. Curr. Opin. Insect Sci. 20, 7–12. https://doi.org/10.1016/j. cois.2017.02.002.

13. Cozzarolo, C.-S., Glazot, O., Christe, P., and Pigeault, R. (2020). Enhanced Attraction of Arthropod Vectors to Infected Vertebrates: A Review of Empirical Evidence. Front. Ecol. Evol. 8, 568140. https://doi.org/10.3389/fevo. 2020.568140.

14. Zhang, H., Zhu, Y., Liu, Z., Peng, Y., Peng, W., Tong, L., Wang, J., Liu, Q., Wang, P., and Cheng, G. (2022). A volatile from the skin microbiota of flavivirus-infected hosts promotes mosquito attractiveness. Cell 185, 2510–2522.e16. https://doi.org/10.1016/j. cell.2022.05.016.

15. Maire, T., Lambrechts, L., and Hol, F.J.H. (2024). Arbovirus impact on mosquito

behavior: the jury is still out. Trends Parasitol. 40, 292–301. https://doi.org/10.1016/j.pt. 2024.02.004.

16. Platt, K.B., Linthicum, K.J., Myint, K.S., Innis, B.L., Lerdthusnee, K., and Vaughn, D.W. (1997). Impact of dengue virus infection on feeding behavior of *Aedes Aegypti*. Am. J. Trop. Med. Hyg. 57, 119–125.

17. Sim, S., Ramirez, J.L., and Dimopoulos, G. (2012). Dengue Virus Infection of the *Aedes aegypti* Salivary Gland and Chemosensory Apparatus Induces Genes that Modulate Infection and Blood-Feeding Behavior. PLoS Pathog. 8, e1002631. https://doi.org/10. 1371/journal.ppat.1002631.

18. Sylvestre, G., Gandini, M., and Maciel-de- Freitas, R. (2013). Age-Dependent Effects of Oral Infection with Dengue Virus on *Aedes aegypti* (Diptera: Culicidae) Feeding Behavior, Survival, Oviposition Success and Fecundity. PLoS One 8, e59933. https://doi. org/10.1371/journal.pone.0059933.

19. Maciel-de-Freitas, R., Sylvestre, G., Gandini, M., and Koella, J.C. (2013). The Influence of Dengue Virus Serotype-2 Infection on *Aedes aegypti* (Diptera: Culicidae) Motivation and Avidity to Blood Feed. PLoS One 8, e65252. https://doi.org/10.1371/journal.pone. 0065252.

20. Wei Xiang, B.W., Saron, W.A.A., Stewart, J.C., Hain, A., Walvekar, V., Missé, D., Thomas, F., Kini, R.M., Roche, B., Claridge-Chang, A., et al. (2022). Dengue virus infection modifies mosquito blood-feeding behavior to increase transmission to the host. Proc. Natl. Acad. Sci. USA 119, e2117589119. https://doi.org/10. 1073/pnas.2117589119.

21. Putnam, J.L., and Scott, T.W. (1995). Blood-Feeding Behavior of Dengue-2 Virus-Infected *Aedes Aegypti*. Am. J. Trop. Med. Hyg. 52,
225-227. https://doi.org/10.4269/ajtmh.1995.52.225.

22. Vasilakis, N., Cardosa, J., Hanley, K.A., Holmes, E.C., and Weaver, S.C. (2011). Fever from the forest: prospects for the continued emergence of sylvatic dengue virus and its impact on public health. Nat. Rev. Microbiol. 9, 532-541. https://doi.org/10.1038/nrmicro2595.

23. Valentine, M.J., Murdock, C.C., and Kelly, P.J. (2019). Sylvatic cycles of arboviruses in non-human primates. Parasit. Vectors 12, 463. https://doi.org/10.1186/s13071-019-3732-0.

24. Vasilakis, N., and Weaver, S.C. (2017). Flavivirus transmission focusing on Zika. Curr. Opin. Virol. 22, 30-35. https://doi.org/10.1016/j.coviro.2016.11.007.

25. Mayer, S.V., Tesh, R.B., and Vasilakis, N. (2017). The emergence of arthropod-borne viral diseases: A global prospective on dengue, chikungunya and zika fevers. Acta Trop. 166, 155-163. https://doi.org/10.1016/j.actatropica.2016.11.020.

26. Hanley, K.A., Monath, T.P., Weaver, S.C., Rossi, S.L., Richman, R.L., and Vasilakis, N. (2013). Fever versus fever: The role of host and vector susceptibility and interspecific competition in shaping the current and future distributions of the sylvatic cycles of dengue virus and yellow fever virus. Infect. Genet. Evol. 19, 292-311. https://doi.org/10.1016/j.meegid.2013.03.008.

27. Gubler, D.J., Vasilakis, N., and Musso, D. (2017). History and Emergence of Zika Virus. J. Infect. Dis. 216, S860-S867. https://doi.org/10.1093/infdis/jix451.

28. Pereira-dos-Santos, T., Roiz, D., Lourenço-de-Oliveira, R., and Paupy, C. (2020). A Systematic Review: Is Aedes albopictus an Efficient Bridge Vector for Zoonotic Arboviruses? Pathogens 9, 266. https://doi.org/10.3390/pathogens9040266.

29. Chua, C.L., Chan, Y.F., Andu, E.S.G.S., Rovier-Yan, J.J., Sitam, F.T., Verasahib, K., and Sam, I.C. (2019). Little Evidence of Zika Virus Infection in Wild Long-Tailed Macaques, Peninsular Malaysia. Emerg. Infect. Dis. 25, 374-376. https://doi.org/10.3201/eid2502.180258.

30. Hanley, K.A., Cecilia, H., Azar, S.R., Moehn, B.A., Gass, J.T., Oliveira da Silva, N.I., Yu, W., Yun, R., Althouse, B.M., Vasilakis, N., and Rossi, S.L. (2024). Trade-offs shaping transmission of sylvatic dengue and Zika viruses in monkey hosts. Nat. Commun. 15, 2682. https://doi.org/10.1038/s41467-024-46810-x.

31. Hernandez Acosta, E. (2023). Some like it Hot: How Urban Microclimate across a Tropical City Impacts the Capacity of Aedes Mosquitoes to Transmit Flaviviruses (New Mexico State University). PhD thesis.

32. Buchta, J.N., Zarndt, B.S., Garver, L.S., Rowland, T., Shi, M., Davidson, S.A., and Rowton, E.D. (2015). Blood-Feeding Behaviors of Anopheles stephensi but not Phlebotomus papatasi are Influenced by Actively Warming Guinea Pigs (Cavia porcellus) Under General Anesthesia. J. Am. Mosq. Control Assoc. 31, 149-154. https://doi.org/10.2987/14-6467.

33. Paupy, C., Delatte, H., Bagny, L., Corbel, V., and Fontenille, D. (2009). Aedes albopictus, an arbovirus vector: From the darkness to the light. Microbes Infect. 11, 1177-1185. https://doi.org/10.1016/j.micinf.2009.05.005.

34. Carnaghi, M., Mandelli, F., Feugère, L., Joiner, J., Young, S., Belmain, S.R., Hopkins, R.J., and Hawkes, F.M. (2024). Visual and

thermal stimuli modulate mosquito-host contact with implications for improving malaria vector control tools. iScience 27, 108578. https://doi.org/10.1016/j.isci.2023.108578.

35. Council, S.E., Savage, A.M., Urban, J.M., Ehlers, M.E., Skene, J.H.P., Platt, M.L., Dunn, R.R., and Horvath, J.E. (2016). Diversity and evolution of the primate skin microbiome. Proc. Biol. Sci. 283, 20152586. https://doi.org/10.1098/rspb.2015.2586.

36. Verhulst, N.O., Umanets, A., Weldegergis, B.T., Maas, J.P.A., Visser, T.M., Dicke, M., Smidt, H., and Takken, W. (2018). Do apes smell like humans? The role of skin bacteria and volatiles of primates in mosquito host selection. J. Exp. Biol. 221, 185959. https://doi.org/10.1242/jeb.185959.

37. Bakker, J.W., Loy, D.E., Takken, W., Hahn, B.H., and Verhulst, N.O. (2020). Attraction of mosquitoes to primate odours and implications for zoonotic Plasmodium transmission. Med. Vet. Entomol. 34, 17-26. https://doi.org/10.1111/mve.12402.

38. McMeniman, C.J., Corfas, R.A., Matthews, B.J., Ritchie, S.A., and Vosshall, L.B. (2014). Multimodal Integration of Carbon Dioxide and Other Sensory Cues Drives Mosquito Attraction to Humans. Cell 156, 1060-1071. https://doi.org/10.1016/j.cell.2013.12.044.

39. Greppi, C., Laursen, W.J., Budelli, G., Chang, E.C., Daniels, A.M., Van Giesen, L., Smidler, A.L., Catteruccia, F., and Garrity, P.A. (2020). Mosquito heat seeking is driven by an ancestral cooling receptor. Science 367, 681-684. https://doi.org/10.1126/science.aay9847.

40. Lindsay, S., Ansell, J., Selman, C., Cox, V., Hamilton, K., and Walraven, G. (2000). Effect of pregnancy on exposure to malaria mosquitoes. Lancet 355, 1972. https://doi.org/10.1016/S0140-6736(00)02334-5.

41. Ellwanger, J.H., Cardoso, J.D.C., and Chies, J.A.B. (2021). Variability in human attractiveness to mosquitoes. Curr. Res. Parasitol. Vector. Borne. Dis. 1, 100058. https://doi.org/10.1016/j.crpvbd.2021.100058.

42. Christofferson, R.C., Wearing, H.J., Turner, E.A., Walsh, C.S., Salje, H., Tran-Kiem, C., and Cauchemez, S. (2022). How do it bite thee? Let me count the ways: Exploring the implications of individual biting habits of Aedes aegypti for dengue transmission. PLoS Negl. Trop. Dis. 16, e0010818. https://doi.org/10.1371/journal.pntd.0010818.

43. Rock, K.S., Wood, D.A., and Keeling, M.J. (2015). Age- and bite-structured models for vector-borne diseases. Epidemics 12, 20-29. https://doi.org/10.1016/j.epidem.2015.02.006.

44. Wu, S.L., Sánchez C, H.M., Henry, J.M., Citron, D.T., Zhang, Q., Compton, K., Liang, B., Verma, A., Cummings, D.A.T., Le Menach, A., et al. (2020). Vector bionomics and vectorial capacity as emergent properties of mosquito behaviors and ecology. PLoS Comput. Biol. 16, e1007446. https://doi.org/10.1371/journal.pcbi.1007446.

45. Mayton, E.H., Tramonte, A.R., Wearing, H.J., and Christofferson, R.C. (2020). Age-structured vectorial capacity reveals timing, not magnitude of within-mosquito dynamics is critical for arbovirus fitness assessment. Parasit. Vectors 13, 310. https://doi.org/10.1186/s13071-020-04181-4.

46. Cecilia, H., Drouin, A., Métras, R., Balenghien, T., Durand, B., Chevalier, V., and Ezanno, P. (2022). Mechanistic models of Rift Valley fever

virus transmission: A systematic review. PLoS Negl. Trop. Dis. 16, e0010339. https://doi.org/10.1371/journal.pntd.0010339.

47. Althouse, B.M., Guerbois, M., Cummings, D.A.T., Diop, O.M., Faye, O., Faye, A., Diallo, D., Sadio, B.D., Sow, A., Faye, O., et al. (2018). Role of monkeys in the sylvatic cycle of chikungunya virus in Senegal. Nat. Commun. 9, 1046. https://doi.org/10.1038/s41467-018-03332-7.

48. Scott, T.W., and Takken, W. (2012). Feeding strategies of anthropophilic mosquitoes result in increased risk of pathogen transmission. Trends Parasitol. 28, 114-121. https://doi.org/10.1016/j.pt.2012.01.001.

49. Zahid, M.H., Van Wyk, H., Morrison, A.C., Coloma, J., Lee, G.O., Cevallos, V., Ponce, P., and Eisenberg, J.N.S. (2023). The biting rate of Aedes aegypti and its variability: A systematic review (1970-2022). PLoS Negl. Trop. Dis. 17, e0010831. https://doi.org/10.1371/journal.pntd.0010831.

50. Armstrong, P.M., Ehrlich, H.Y., Magalhaes, T., Miller, M.R., Conway, P.J., Bransfield, A., Misencik, M.J., Gloria-Soria, A., Warren, J.L., Andreadis, T.G., et al. (2020). Successive blood meals enhance virus dissemination within mosquitoes and increase transmission potential. Nat. Microbiol. 5, 239-247. https://doi.org/10.1038/s41564-019-0619-y.

51. Brackney, D.E., LaReau, J.C., and Smith, R.C. (2021). Frequency matters: How successive feeding episodes by blood-feeding insect vectors influences disease transmission. PLoS Pathog. 17, e1009590. https://doi.org/10.1371/journal.ppat.1009590.

52. Johnson, R.M., Cozens, D.W., Ferdous, Z., Armstrong, P.M., and Brackney, D.E. (2023). Increased blood meal size and feeding frequency compromise Aedes aegypti midgut integrity and enhance dengue virus dissemination. PLoS Negl. Trop. Dis. 17, e0011703. https://doi.org/10.1371/journal.pntd.0011703.

53. Roche, B., and Guégan, J.-F. (2011). Ecosystem dynamics, biological diversity and emerging infectious diseases. C. R. Biol. 334, 385-392. https://doi.org/10.1016/j.crvl.2011.02.008.

54. Gandon, S. (2018). Evolution and Manipulation of Vector Host Choice. Am. Nat. 192, 23-34. https://doi.org/10.1086/697575.

55. Vogt, M.B., Lahon, A., Arya, R.P., Kneubehl, A.R., Spencer Clinton, J.L., Paust, S., and Rico-Hesse, R. (2018). Mosquito saliva alone has profound effects on the human immune system. PLoS Negl. Trop. Dis. 12, e0006439. https://doi.org/10.1371/journal.pntd.0006439.

56. Schneider, B.S., and Higgs, S. (2008). The enhancement of arbovirus transmission and disease by mosquito saliva is associated with modulation of the host immune response. Trans. R. Soc. Trop. Med. Hyg. 102, 400-408. https://doi.org/10.1016/j.trstmh.2008.01.024.

57. McCracken, M.K., Gromowski, G.D., Garver, L.S., Goupil, B.A., Walker, K.D., Friberg, H., Currier, J.R., Rutvisuttinunt, W., Hinton, K.L., Christofferson, R.C., et al. (2020). Route of inoculation and mosquito vector exposure modulate dengue virus replication kinetics and immune responses in rhesus macaques. PLoS Negl. Trop. Dis. 14, e0008191. https://doi.org/10.1371/journal.pntd.0008191.

58. Visser, I., Koenraadt, C.J.M., Koopmans, M.P.G., and Rockx, B. (2023). The significance of mosquito saliva in arbovirus transmission and pathogenesis in the vertebrate host. One
Health 16, 100506. https://doi.org/10.1016/j. onehlt.2023.100506.

59. Guerrero, D., Cantaert, T., and Missé, D. (2020). Aedes Mosquito Salivary Components and Their Effect on the Immune Response to Arboviruses. Front. Cell. Infect. Microbiol. 10, 407. https://doi.org/10.3389/fcimb.2020. 00407.

60. Manning, J.E., Morens, D.M., Kamhawi, S., Valenzuela, J.G., and Memoli, M. (2018). Mosquito Saliva: The Hope for a Universal Arbovirus Vaccine? J. Infect. Dis. 218, 7–15. https://doi.org/10.1093/infdis/jiy179.

61. Guerrero, D., Vo, H.T.M., Lon, C., Bohl, J.A., Nhik, S., Chea, S., Man, S., Sreng, S., Pacheco, A.R., Ly, S., et al. (2022). Evaluation of cutaneous immune response in a controlled

human in vivo model of mosquito bites. Nat. Commun. 13, 7036. https://doi.org/10.1038/ s41467-022-34534-9.

62. Osada, N., Kohara, A., Yamaji, T., Hirayama, N., Kasai, F., Sekizuka, T., Kuroda, M., and Hanada, K. (2014). The Genome Landscape of the African Green Monkey Kidney-Derived Vero Cell Line. DNA Res. 21, 673–683. https:// doi.org/10.1093/dnares/dsu029.

63. Miller, J.R., Koren, S., Dilley, K.A., Puri, V., Brown, D.M., Harkins, D.M., Thibaud-Nissen, F., Rosen, B., Chen, X.G., Tu, Z., et al. (2018). Analysis of the Aedes albopictus C6/36 genome provides insight into cell line utility for viral propagation. GigaScience 7, 1–13. https://doi.org/10.1093/gigscience/gix135.

64. Benjamini, Y., and Hochberg, Y. (1995). Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J. Roy. Stat. Soc. B 57, 289–300. https://doi.org/10.1111/j.2517-6161.1995. tb02031.x.

65. Hendy, A., Hernandez-Acosta, E., Valério, D., Fé, N.F., Mendonça, C.R., Costa, E.R., Andrade, E.S.d., Andes Júnior, J.T., Assunção, F.P., Scarpassa, V.M., et al. (2023). Where boundaries become bridges: Mosquito community composition, key vectors, and environmental associations at forest edges in the central Brazilian Amazon. PLoS Negl Trop Dis 17, e0011296. https://doi.org/10.1371/journal.pntd. 0011296.
## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td><strong>Antibodies</strong></td><td></td><td></td></tr><tr><td>mouse hyperimmune serum against DENV-2 NGC</td><td>World Reference Center on Emerging Viruses and Arboviruses (WRCEVA) at University of Texas Medical Branch (UTMB)</td><td>MIAF T-35432, generated 12/10/2014</td></tr><tr><td>mouse hyperimmune serum against ZIKV strain MR-766</td><td>World Reference Center on Emerging Viruses and Arboviruses (WRCEVA) at University of Texas Medical Branch (UTMB)</td><td>MIAF T-36846 generated 07/19/2017</td></tr><tr><td><strong>Bacterial and virus strains</strong></td><td></td><td></td></tr><tr><td>ylvatic DENV-2</td><td>World Reference Center on Emerging Viruses and Arboviruses (WRCEVA) at University of Texas Medical Branch (UTMB)</td><td>strain P8-1407</td></tr><tr><td>ylvatic ZIKV</td><td>World Reference Center on Emerging Viruses and Arboviruses (WRCEVA) at University of Texas Medical Branch (UTMB)</td><td>strain DakAR 41525</td></tr><tr><td><strong>Critical commercial assays</strong></td><td></td><td></td></tr><tr><td>Cytokine 29-Plex Monkey Panel</td><td>ThermoFisher Scientific, Waltham, MA, USA</td><td>N/A</td></tr><tr><td><strong>Deposited data</strong></td><td></td><td></td></tr><tr><td>Raw and analyzed data</td><td>This paper; Zenodo</td><td>https://doi.org/10.5281/zenodo.12543011</td></tr><tr><td><strong>Experimental models: cell lines</strong></td><td></td><td></td></tr><tr><td>African green monkey kidney cells (Vero)</td><td>American Type Culture Collection (ATCC, Bethesda, MD, USA)</td><td>catalog number CCL-81</td></tr><tr><td>larval Ae. albopictus cells (C6/36)</td><td>American Type Culture Collection (ATCC, Bethesda, MD, USA)</td><td>catalog number CRL-1660</td></tr><tr><td><strong>Experimental models: organisms/strains</strong></td><td></td><td></td></tr><tr><td>Aedes albopictus</td><td>Galveston Texas 2018</td><td>N/A</td></tr><tr><td>Cynomolgus macaques (Macaca fascicularis)</td><td>Worldwide Primates, Inc (Miami, FL, USA)</td><td>N/A</td></tr><tr><td>Black capped squirrel monkeys (Saimiri boliviensis boliviensis)</td><td>MD Anderson Center (Bastrop, TX, USA)</td><td>N/A</td></tr><tr><td><strong>Software and algorithms</strong></td><td></td><td></td></tr><tr><td>R software (version 4.3.1)</td><td>R Foundation</td><td>https://www.r-project.org/foundation/</td></tr><tr><td>Rstudio (version 2023.06.1, "Mountain Hydrangea" Release)</td><td>Posit</td><td>https://posit.co/products/open-source/rstudio/</td></tr><tr><td>Code for data analysis</td><td>Zenodo</td><td>https://doi.org/10.5281/zenodo.12543011</td></tr></tbody></table>
# EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

## Non-human primates (NHPs)

Sixteen (9 females, 7 males) Mauritius origin cynomolgus macaques (Macaca fascicularis) of both sexes, between 2.2 and 4.0 years of age and weighing between 2.7 and 5.2 kg at the time of the study, were purchased from Worldwide Primates, Inc (Miami, FL, USA). Twenty-four (12 females, 12 males) squirrel monkeys (Saimiri boliviensis boliviensis) of both sexes, between 4 and 14 years of age and weighing between 0.62 kg and 0.89 kg at the time of the study, were purchased from the MD Anderson Center (Bastrop, TX, USA). All NHPs were anesthetized with ketamine before every procedure. Temperature for NHP maintenance was 26-28 °C for squirrel monkeys and 24-26 °C for cynomolgus macaques. NHPs were maintained on a 12:12 h light:dark cycle, with lights on from 7a.m. to 7p.m. Macaques were single housed and squirrel monkeys were pair housed, in open metal caging that allowed for visual but not physical contact with other animals in the room. Standard primate chow was provided twice daily, with enrichment in the form of fruits and vegetables added once daily. All animals received a minimum of twice daily health checks. The experiments conducted here were approved via UTMB Institutional Animal Care and Use Committee (IACUC) protocol 1912100.

## Mosquitoes

Adult Ae. albopictus (Galveston, 2018) mosquitoes were maintained at 28°C while the temperature of the rearing room (from larval development to adult emergence) varied between 26 and 28°C. Mosquitoes used to infect NHPs were F12 for macaques and F14 for squirrel monkeys.

## Cell lines and virus strains

African green monkey kidney cells (Vero, female,⁶² catalog number CCL-81) and larval Ae. albopictus cells (C6/36, male,⁶³ catalog number CRL-1660) were purchased from the American Type Culture Collection (ATCC, Bethesda, MD, USA). Vero cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 5% (v/v) heat-inactivated fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch, GA, USA), 1% (v/v) Penicillin-Streptomycin (P/S, ThermoFisher Scientific, Waltham, MA, USA; 100 U/mL and 100 g/mL, respectively) in a humidified 37°C incubator with 5% CO₂. C6/36 cells were maintained in DMEM supplemented with 10% (v/v) heat-inactivated FBS, 10% (v/v) tryptose phosphate broth (TBP, Sigma-Aldrich, St. Louis, MO, USA), 1% (v/v) P/S in a humidified 28°C incubator with 5% CO₂. Vero and C6/36 cells are authenticated approximately yearly via sequencing of COI and comparison with published sequences of African green monkeys and Ae. albopictus, respectively.

The following viruses with the following passage histories were used to infect monkeys: sylvatic DENV-2 strain P8-1407 (suckling mouse 3 passages, C6/36 5 passages) and sylvatic ZIKV strain DakAR 41525 (AP-61 1 passage, C6/36 2 passages, Vero 6 passages).

# METHOD DETAILS

The current study builds upon the experiment described in Hanley et al.³⁰ The experimental design and a detailed sampling scheme are presented in Figure 1.

## Infection of mosquitoes and monkeys

To infect mosquitoes, female Galveston strain Ae. albopictus were intrathoracically inoculated with either a sylvatic Malaysian strain of DENV-2 or a sylvatic African strain of ZIKV and held in screen-top, cardboard cartons (diameter 8 cm, height 8.5 cm) for 10 days. Control mosquitoes were not intrathoracically inoculated but were placed into screen-top, cardboard cartons. To infect monkeys on Day 0 of the experiment, cartons of mosquitoes were placed upon the ear of an anesthetized monkey. Monkeys were exposed to cartons containing a designated number of mosquitoes as follows: Macaques were exposed to cartons containing either 1 (low exposure) or 10 (high exposure) DENV-2-inoculated mosquitoes or 15 ZIKV-inoculated mosquitoes or 10 control mosquitoes. Squirrel monkeys were exposed to cartons containing either 15 DENV-2-inoculated mosquitoes or 15 ZIKV-inoculated mosquitoes or 15 control mosquitoes. Mosquitoes were placed upon the ear of macaques for an average of 7.5 ± 0.62 min (mean ± 1 SE, range = 4 to 10 min) and on the ear of squirrel monkeys for an average of 6.5 ± 0.29 min (mean ± 1 SE, range = 4 to 9 min). Additionally, to test whether non-viremic transmission might occur by long distance movement by arterial blood, uninfected mosquitoes (n = 15) were allowed to feed on squirrel monkeys immediately after they had been fed upon by exposed mosquitoes, on the ear not used for the original feeding. These are hereafter termed "concurrently feeding mosquitoes". Concurrently feeding mosquitoes were held upon the ear for an average of 4.9 ± 0.22 min (mean ± 1 SE, range = 3 to 6 min).

Cartons were then returned to an arthropod containment insectary space, mosquitoes were cold anesthetized, and visibly engorged (partial or full) mosquitoes were separated, counted, and returned to an incubator, while unengorged mosquitoes were counted and discarded. For the purposes of this study, only visibly engorged mosquitoes are considered to have fed. For analyses, the engorgement rate was defined as the ratio of the number of engorged mosquitoes over the total number of mosquitoes in the carton. All raw data on mosquito engorgement on NHPs are provided in Table S1. Two days later, engorged mosquitoes were force-salivated to estimate dose delivered to each monkey via serial dilution and immunostaining on C6/36 cells: 75% [0–100] (mean [min; max]) of the singleton low-exposure DENV-2-inoculated mosquitoes fed on macaques salivated virus, while 54% [38–86] of high-exposure DENV-2-inoculated mosquitoes that engorged on macaques, 88% [71–100] of ZIKV-inoculated mosquitoes that engorged on macaques, 77% [40–100] of DENV-2-inoculated mosquitoes that engorged on squirrel monkeys, and 83% [20–100] of ZIKV-inoculated mosquitoes that engorged on squirrel monkeys salivated virus.
<figure><img src="image_15.png" /></figure>

## Monitoring viremia, cytokine responses, and transmission

For both host species, blood was drawn at day −7 and at designated intervals from day 1–28 after the initial exposure to mosquitoes to quantify infectious viremia via serial dilution and immunostaining³⁰ and cytokine concentrations (Figure 1B). Clarified serum from each animal was subjected to the Cytokine 29-Plex Monkey Panel (ThermoFisher Scientific, Waltham, MA, USA), measuring TGF-β, G-CSF, RANTES, Eotaxin, MIP-1β, GM-CSF, MIP-1β, MCP-1, HGF, VEGF, IFN-γ, MDC, I-TAC, MIF, TNF-α, IP-10, MIG, and interleukins (IL) 1β, 1RA, 2, 4, 6, 8, 10, 12, 15, 17. Each sample was run in duplicate according to manufacturer instructions and average values were ascertained via the standard curve generated for each cytokine per run. Weight was measured each time an NHP was handled. Due to their small size, squirrel monkeys were assigned to one of two cohorts, sampled on different days (Figure 1B). All NHPs were surgically implanted with DST micro-T temperature loggers (Star-Oddi, Garðabær, Iceland) set to record temperature every 15 min. Host body temperature at the time of mosquito feeding was linearly interpolated using available temperature records. Pre-study blood samples were screened via PRNT assays against a panel of flaviviruses and were all negative.³⁰

To assess transmission from infected hosts to mosquitoes, for both species, cartons of approximately 10 (range 9–12, for DENV-2-infected and control macaques) or 15 (range 15–22, for all other treatments) five-to seven-day-old, uninfected Ae. albopictus that had been starved of sucrose for 24 h were allowed to feed on NHP ears on the days designated in Figure 1B, and the time of day and duration of exposure were monitored. In two cases, the end time of exposure was not recorded, and instead we used the time the NHP was returned to its cage, which is a slight overestimation (Table S1). The ear used for feeding was alternated at each feeding day. Engorged mosquitoes were separated as described above and incubated for 14 days, after which bodies and legs were separated and assayed for virus infection.³⁰ In some cases, NHPs with no detectable viremia, even after sera were passaged in Vero cells, nonetheless transmitted virus to mosquitoes. In these cases, we assigned a viremia of 10 PFU/mL (half the limit of detection) to the monkey. Similarly, if an NHP was not detectably viremic nor transmitted to mosquitoes on a given sampling day, but was viremic and/or transmitted to mosquitoes on the previous and following sampling days, we also assigned a viremia of 10 PFU/mL. The remaining samples with no detectable viremia even after passage, and no transmission to mosquitoes were considered as true absence of viremia.

Squirrel monkeys were euthanized at the termination of this experiment but cynomolgus macaques were not. On day 28 of the experiment, squirrel monkeys were being prepared for euthanasia, which necessitated some changes in the way they were handled, and induced important differences with cynomolgus macaques with regards to time of day and duration of mosquito exposure, which could bias our analyses. Indeed, on day 28, mosquito exposure happened significantly later than on other days in squirrel monkeys ($p < 2 \times 10^{-16}$), but not in cynomolgus macaques ($p = 0.12$). On day 28, exposure also lasted significantly longer than on other days in squirrel monkeys (8–15 min, with a median of 11 min for day 28, and 4 to 12 min, with a median of 6 min for other days, $p = 1.7 \times 10^{-9}$), but not in cynomolgus macaques (5–8 min, with a median of 6 min for day 28, and 3 to 16 min, with a median of 6 min for other days, $p = 0.77$). To avoid skewing the distribution of these time metrics, we excluded data on day 28 of the experiment, for both species (from $n = 331$ to $n = 293$). In our final dataset, mosquito exposure events happened between 8:37 a.m. and 2:32 p.m.

## QUANTIFICATION AND STATISTICAL ANALYSIS

### Engorgement and mosquito viral inoculation

We tested whether the virus inoculation status of mosquitoes affected their engorgement rate on day 0 of the experiments. As none of the concurrently feeding mosquitoes became infected (Table S2), we aggregated the concurrently feeding and control mosquitoes. We excluded mosquitoes belonging to the low exposure group (1 mosquito per NHP) feeding on cynomolgus macaques, as they all engorged. We also tested differences in engorgement per host species for a given mosquito status (control, DENV-2 or ZIKV inoculated). Model selection focused on choosing between binomial and betabinomial error distributions (both with a logit link function), the latter accounting for dispersion anomalies, as well as between a generalized linear model (fixed effects only) and a generalized mixed effect model, incorporating monkey ID as a random effect (intercept). Duration of mosquito exposure was used as an offset, i.e., a scaling factor fixed at 1 reflecting that the tendency to engorge is influenced by the duration of exposure to the host. The models were fitted using maximum likelihood and the selection was done through inspection of models' residuals, likelihood ratio tests, and corrected Akaike Information Criterion (AICc) comparison. Comparisons of groups were made using posthoc Wald tests (also called z-test). The values of *n* per group can be found in the legend of Figure 2. We confirmed that significant results remained as such after using the Benjamini-Hochberg correction for *p* values, with a false discovery rate (false positives/(false positives + true positives)) of 5%,⁶⁴ but we report the unadjusted *p* values in the results section. When showing predicted probabilities of engorgement from the model, we assume a common duration of 6 min, corresponding to the median of all durations of exposure to the hosts.

### Preliminary observations

We performed tests to decide on the inclusion of interaction terms in our complete model (described in the next section). Data for days 0 and 28 were excluded. Tests on temperature were performed using a generalized linear mixed effect model with monkey ID and day as random intercepts. Tests on weights used a linear model, and tests on duration of exposure used a generalized linear model with a Poisson error distribution.

Over the whole course of the experiments, temperatures were lower in cynomolgus macaques than in squirrel monkeys (Figure S2), both in control ($p = 0.002$), DENV-2 infected NHPs ($p = 0.0004$), and ZIKV-infected animals ($p = 0.001$). This species difference was still apparent when
restricting to temperatures at the time of mosquito exposure, and in addition, temperatures were higher in DENV-2 infected than control cynomolgus macaques (Figure S2, *p* = 0.022). Still, temperatures of all individuals remained within the normal temperature ranges expected for each species ([36, 39.5] °C for cynomolgus macaques and [33.3, 41] °C for squirrel monkeys). Besides, the time at which mosquito exposure happened influenced host temperature (Figure S2). In cynomolgus macaques, we noticed signs of hypothermia (Figures S2A and S2B), likely due to anesthesia, and because of the absence of heating blanket, but mosquito exposure mostly happened before such hypothermia (Figures S2A and S2B). We also noted that within monkey species, the variability of temperature at the time of mosquito exposure was limited to a range of about 2 °C (Figure S2). As a result of these observations, we decided to include an interaction term between temperature and species in our complete model of mosquito engorgement on days 1–21.

Squirrel monkeys were lighter than cynomolgus macaques (*p* < 2 e−16, see Hanley et al.³⁰). We therefore included an interaction term between weight and species in our complete model of mosquito engorgement on days 1–21.

The duration of mosquito exposure lasted on average longer on ZIKV-infected macaques than control cynomolgus macaques (6 min [5, 7] for controls, 8 min [7, 9] for ZIKV-infected, *p* = 0.0008). This did not result in the inclusion of an interaction term in our complete model as duration was taken into account as an offset.

## Engorgement and host infection

We tested whether the infection status of NHPs affected uninfected mosquitoes' engorgement rates. We started with a simple model comparing the experimental groups (combination of NHP species and infection status, and virus species) to see what could be concluded in the absence of other information. Then, to account for heterogeneity within these groups, we built a complete model, accounting for virus species, time of day, host sex, species, body temperature at the time of feeding, weight, and viremia. Interactions between host species and weight, as well as between host species and host body temperature, were included. All quantitative variables were scaled except viremia. Duration of mosquito exposure was used as an offset in both the simple and complete models. The model selection procedure was similar to the one described earlier, except that generalized mixed effect models also included days post infection as random intercepts. When showing predicted probabilities of engorgement from the model, we assume a common duration of 6 min, corresponding to the median of all durations of exposure to the hosts. The values of *n* per group can be found in the legend of Figure 3.

## Comparison with free-living *Aedes albopictus*

In order to compare the inter-individual heterogeneity in our experimental dataset to heterogeneity in the approach rate of free-living mosquitoes, we took advantage of an experiment designed to test the impact of levels of urbanization on the approach of *Aedes* spp. mosquitoes to humans.³¹ In this experiment, human approach rate was measured by handheld net collections of mosquitoes that would approach within an arm's length to the collector. Collectors were wearing mosquito repellent to prevent bites.

Collection of approach data in Hernandez-Acosta 2023³¹ was done across three levels of urbanization, at the following collection windows: 6:00-7:00, 9:00-10:00, 12:00-13:00, 15:00-16:00, and 18:00-19:00 h. Urbanization was measured by the Normalized Difference Built-up Index (NDBI) and classified using the Jenks natural breaks method into low (−1 to −0.467), medium (−0.468 to −0.098), and high (−0.099 to 1.000) NDBI value categories.⁶⁵ From this larger dataset, we extracted the number of approaches per collector at times and at urbanization levels showing the highest number of female *Ae. albopictus* approaching, as well as the highest proportion of non-zero approach among attempts. These optimal conditions were observed for low and medium NDBI, and for collection windows 12:00-13:00 and 15:00-16:00.

To compare experimental and field data, we used the coefficient of variation (standard deviation/mean), which is unitless. We computed it on engorgement rates normalized by duration of exposure, per NHP, for our dataset, and on number of approached females *Ae. albopictus*, per collector (the duration of collection being constant), in optimal conditions defined above, for approach data.

## Repeated mosquito bites and cytokine response

We assessed the effect of repeated exposure to uninfected mosquitoes on control NHP cytokine concentration (*n* = 4 control cynomolgus macaques and 4 control squirrel monkeys). We used log₁₀ cytokine concentration as the response variable, and variables measuring mosquito exposure as fixed effects (Table S3). For simplicity, here we use the term bite to refer to the number of engorged mosquitoes at the end of the exposure period, although we did not observe the number of biting events per se. We considered the effects of biting in the short term (the number of bites received the day prior for cynomolgus macaques, and 2 days prior for squirrel monkeys, due to alternating days of exposure) and the long term (the cumulative number of bites received in the last seven days for both species).

We selected between linear and generalized additive models using AICc. Competing models were fitted using maximum likelihood, for each monkey species and each cytokine separately. Monkey ID was used as a random effect (intercept). Day was not used as it did not improve AICc. In the additive model, the number of knots was constrained to 4 for fixed effects and to 2 for random intercepts, to avoid over-fitting. All concentrations below the limit of detection (LOD) were excluded from analysis initially, for all models. This substantially improved the residuals of the models, which were checked using the R package DHARMa. For each model type, we checked that significant results remained as such after using the Benjamini-Hochberg correction for *p* values, with a false discovery rate (false positives/(false positives + true positives)) of 5%,⁶⁴ but we report the initial *p* values in the results section. For the selected model (linear or additive), if a result stood out after *p* value correction, the same model including measures below LOD (in that case, fixed at LOD) was run and compared. The concurrent change of short-term and long-term bite exposure is shown in Figure S4, for each control cynomolgus macaque, as this is the only species where significant effects were detected.