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Cell Rep
Cell Rep
Cell reports
2211-1247

39126653
10.1016/j.celrep.2024.114600
nihpa2019540
Article
Anopheles gambiae lacking AgTRIO probe inefficiently on a mammalian host
Chuang Yu-Min 145*
Dong Yuemei 24
Stone Helen 1
Abouneameh Selma 1
Tang Xu-Dong 3
Raduwan Hamidah 1
Dimopoulos George 2
Fikrig Erol 1
1 Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA
2 W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA
3 Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China
4 These authors contributed equally
5 Lead contact
AUTHOR CONTRIBUTIONS

Conceptualization, Y.-M.C., G.D., and E.F.; methodology, data curation, project administration, and formal analysis, Y.-M.C., H.S., Y.D., H.R.., S.A., and X.-D.T.; funding acquisition, E.F.; writing– original draft, Y.-M.C.; writing – review and editing, Y.-M.C., Y.D., G.D., and E.F.

* Correspondence: yu-min.chuang@yale.edu
11 9 2024
27 8 2024
08 8 2024
17 9 2024
43 8 114600114600
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/).
SUMMARY

Malaria is initiated as Plasmodium sporozoites are injected into the dermis when an infected mosquito probes on a vertebrate host for a blood meal. Factors in the mosquito saliva, such as AgTRIO, can alter the ability of Anopheles gambiae to transmit Plasmodium. We therefore used CRISPR-Cas9-mediated genome editing to generate AgTRIO knockout (KO) A. gambiae and examined the ability of these mosquitoes to probe on a vertebrate host. AgTRIO KO mosquitoes showed a diminished host probing capacity and required repetitive probing to locate a blood resource to complete a blood meal. This increased probing resulted in enhanced Plasmodium transmission to the vertebrate host. Our data demonstrate the importance of the A. gambiae saliva protein AgTRIO in probing and its influence on the ability of mosquitoes to transmit malaria.

In brief

Chuang et al. use CRISPR-Cas9 to generate AgTRIO knockout mosquitoes and demonstrate that AgTRIO is required for mosquitoes to probe efficiently during a blood meal. Lacking AgTRIO causes mosquitoes to take longer when probing to take a blood meal. This increased probing results in enhanced Plasmodium transmission to the vertebrate host.

Graphical abstract
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pmcINTRODUCTION

Hematophagous arthropods rely on blood to produce eggs. Components of the blood meal provide nutrients for protein synthesis that are important for ovarian function. Arthropod-borne pathogens, including Plasmodium, that cause malaria, co-opt the blood-feeding process. When Anopheles gambiae feed on a Plasmodium-infected host, Plasmodium is ingested by mosquitoes and completes the sporogonic portion of its life cycle inside mosquitoes. After development in the mosquito midgut, sporozoite-stage parasites invade the salivary glands from where they are injected into the dermis of the vertebrate host during the blood meal. The ability of a mosquito to acquire a blood meal is a critical bottleneck in the Plasmodium life cycle.

To obtain a blood meal, A. gambiae land on the hosts’ skin, pierce through the epidermis, and probe the dermis to search for microcapillaries from which they can engorge.1 Mosquitoes may need to probe multiple times to find a blood microcapillary, and during the probing process, saliva is secreted into the dermis to facilitate blood feeding. Plasmodium sporozoites are injected with the saliva into the tissue during probing.2 Mosquito saliva facilitates the engorgement process by increasing vascular permeability, preventing coagulation, and altering the bite-site microenvironment.2–7 Mosquito saliva has been linked to probing.8–10 As an example, silencing SG6, which encodes an Anopheles salivary gland protein, alters probing and blood feeding.11 Plasmodium sporozoites have intimate interactions with mosquito saliva components in the salivary glands and during migration to the dermis of the vertebrate host.2,12–16 Indeed, the immune response against one of the salivary gland proteins, AgTRIO, contributes to reducing the Plasmodium burden during early infection of mice.16–18 Consistent with this, RNAi-mediated silencing of AgTRIO in A. gambiae affects Plasmodium transmission in the skin.14 These data indicate that AgTRIO plays an important role during Plasmodium transmission to the mammalian host. We therefore used the CRISPR-Cas9 system to generate AgTRIO knockout (KO) A. gambiae and examined the ability of these mosquitoes to probe on a host, take a blood meal, and transmit Plasmodium parasites.

RESULTS

Generation of AgTRIO KO A. gambiae

To better understand the role of AgTRIO during mosquito probing, feeding, and Plasmodium transmission, we used the CRISPR-Cas9 system to generate AgTRIO KO A. gambiae (Figure 1A). A construct with three guide RNAs (gRNAs) targeting AgTRIO was microinjected into A. gambiae docking X1-line embryos to generate an AgTRIO gRNA strain.19 After crossing with X1 reference mosquitoes to eliminate unexpected mutations, the homozygous AgTRIO gRNA strain was crossed with the Vasa-Cas9 strain to generate AgTRIO KO A. gambiae mosquitoes. The sibling strain containing the intact AgTRIO coding sequence was used as the control mosquito. To confirm that AgTRIO was knocked out, individual whole-mosquito genomic DNA was collected and amplified using AgTRIO-specific primers. The control mosquitoes and heterozygous and homozygous AgTRIO mutant mosquitoes were noted by different PCR products on gel electrophoresis (Figure 1B). As AgTRIO is highly expressed in mosquito saliva,16 we also collected salivary gland extracts from control and homozygous AgTRIO KO mosquitoes. There was no AgTRIO expression in the AgTRIO KO mosquitoes, as determined by immunoblots (Figure 1C). Using the CRISPR-Cas9 system, AgTRIO KO A. gambiae were generated successfully.

AgTRIO KO A. gambiae do not probe efficiently on a vertebrate host

AgTRIO is highly expressed in A. gambiae saliva, and it is secreted during the blood meal.14,16 We therefore determined whether AgTRIO contributes to the probing process when mosquitoes search for blood. The time between when a mosquito inserts a mouthpart into a host and a visible blood meal inside the midgut was recorded as the probing time.9,11 Control, gRNA strain, and AgTRIO KO mosquitoes were allowed to feed on mice for up to 10 min. AgTRIO KO A. gambiae probed for a significantly longer period compared to the controls (Figure 2A). AgTRIO KO A. gambiae also required more probing events to complete the blood meal compared to control mosquitoes (Figure 2B). Since the probing process is not required when a blood meal is obtained through an artificial membrane, we let mosquitoes take a blood meal through an artificial membrane feeder. There was no difference between the AgTRIO KO and control groups (Figure 2C). These studies demonstrate that AgTRIO is required for optimal probing by A. gambiae on mice.

We then examined whether the lack of AgTRIO influenced the amount of blood a mosquito could ingest. AgTRIO KO and control A. gambiae were allowed to fully feed on mice, and the amount of ingested blood was determined using colorimetric assays.20 AgTRIO KO A. gambiae contained significantly more blood than the control mosquitoes after the feeding process (Figure 3A). The body length of the control (4.3 ± 0.14 mm, mean ± standard deviation, n = 35) and AgTRIO KO (4.38 ± 0.21 mm, n = 35) mosquitoes were similar, which indicates that the difference in blood intake is not cause by the difference in body size. Since a difference in probing was noted on mice but not membrane feeding, we also determined whether there was a difference in the amount of ingested blood after membrane feeding. While all mosquitoes ingested more blood after membrane feeding compared to feeding on mice, there was no difference between AgTRIO KO and control A. gambiae (Figure 3B).

Lacking AgTRIO does not affect vascular leakage

Increased vascular permeability is an important event that leads to the intake of blood following probing.21 We therefore determined whether the absence of AgTRIO affects vascular leakage at the bite site. We allowed AgTRIO KO or control mosquitoes to feed on the belly of mice that had previously been injected with Evans blue dye. One hour after feeding, the bite sites were removed by punch biopsy, and vascular leakage was determined by the amount of detectable Evans blue dye at the bite site. There was no difference in vascular leakage between the groups (Figure 3C). In addition, cytokine release at the bite site can influence vascular permeability.21 There was no difference in the levels of measurable cytokines at the bite site of AgTRIO KO and control mosquitoes (Figure 3D).

AgTRIO KO A. gambiae transmit more P. berghei to mice

We have demonstrated previously that immunization of mice against AgTRIO could reduce transmission of Plasmodium from mosquito to the host.16–18 Moreover, mosquitoes in which AgTRIO was silenced using RNAi had a normal Plasmodium burden in the salivary glands but transmitted Plasmodium less efficiently to mice.14 We now examined how efficiently AgTRIO KO A. gambiae, which completely lack the gene, transmit Plasmodium to mice. First, we let AgTRIO KO or control mosquitoes take a blood meal from P. berghei-infected mice. After 2 and 3 weeks, the salivary glands of mosquitoes were collected, and the Plasmodium burden was determined by RT-PCR. There was no difference between AgTRIO KO and control A. gambiae (Figure 4A), indicating that AgTRIO does not affect sporozoite invasion of the salivary glands. Next, we collected 300 sporozoites from AgTRIO KO or control mosquitoes and intradermally injected the sporozoites into the ears of C57BL/6 mice. 40 hours later, the livers were dissected, and the Plasmodium infection level was determined by RT-PCR. The Plasmodium liver burden was lower when using sporozoites from AgTRIO KO mosquitoes, supporting the previous observation that AgTRIO contributes to optimal transmission of sporozoites to the host (Figure 4B).14

Next, we performed a mosquito feeding-based infection assay to test for any significant changes in early Plasmodium infection after mosquito bites. We allowed three P. berghei-infected AgTRIO KO or control mosquitoes to obtain blood meals from mice, and the Plasmodium liver burden was then determined by RT-PCR. In contrast to the challenge by intradermal injection, exposure to infected AgTRIO KO mosquitoes resulted in a significantly higher Plasmodium liver burden compared to the control mosquitoes (Figure 4C).

The AgTRIO KO mosquitoes exhibited a longer probing period prior to obtaining blood compared to the controls, a distinction not observed when sporozoites were removed from the mosquitoes and injected directly into animals. These studies indicate that AgTRIO plays a role in A. gambiae mosquito probing behavior, with excessive probing linked to the delivery of more sporozoites into the host.

DISCUSSION

AgTRIO KO mosquitoes, generated using the CRISPR-Cas9 system, offer an ideal model for investigating the importance of AgTRIO throughout the entire process of mosquito feeding on a vertebrate host. Our data demonstrate that the absence of AgTRIO leads to prolonged and less efficient probing by mosquitoes as they attempt to obtain a blood meal. Our membrane feeding studies, where probing is not required, demonstrate that AgTRIO does not play a role in engorgement or other aspects of feeding. While it is beneficial for mosquitoes to probe rapidly and thereby avoid detection by the host, the longer probing time can increase pathogen transmission.22,23 Our results reveal that P. berghei is transmitted to a vertebrate host at a higher rate by AgTRIO KO mosquitoes, indicating that the repeated probing and intermittent deposition of Plasmodium contributes to increased infectivity.

Mosquito probing is a complex and multifaceted process that is not well understood.9–11,24–27 As an example, the D7 family of saliva proteins can function as leukotriene scavengers and therefore interfere with probing.10 Additionally, the salivary protein LIPS-2 has been shown to alter the morphology of the mouthpart protein Cp19, resulting in inefficient mechanical probing.27 Although the mechanism by which AgTRIO influences probing remains to be fully elucidated, it is evident that it does not involve modifications in vascular leakage or local cytokine production. Furthermore, there was no discernible difference in the Plasmodium burden within the salivary glands of AgTRIO KO mosquitoes compared to controls that could have altered pathogen infectivity in the host.

Overall, our findings demonstrate that AgTRIO-KO mosquitoes exhibit a prolonged and repeated probing period before obtaining a blood meal. This extended probing duration correlates with an elevated level of Plasmodium transmission, providing further support the theory that probing is a critical event during which sporozoites are injected into the mammalian host to initiate infection. Understanding the multifactorial influences that modulate A. gambiae probing could lead to novel strategies to influence the capacity of A. gambiae to transmit Plasmodium.

Limitations of the study

There are several limitations of this study. AgTRIO KO mosquitoes could potentially alter the expression of other genes due to the lack of AgTRIO, which may influence the change in probing behavior. Differences when AgTRIO KO and the control mosquitoes take a blood meal under natural feeding conditions may be distinct from a laboratory setting. Moreover, our membrane feeding studies, while important for analyzing the absence of probing, could potentially introduce additional factors that influence the intake of a blood meal. While no differences in vascular permeability or cytokine changes were observed at the bite site, it is possible that our assays did not capture every possible interaction during the short observation period. There might be other mechanisms, besides probing, that contribute to the ability of AgTRIO KO mosquitoes to influence Plasmodium transmission after mosquito bites. Finally, the result with P. berghei transmission may not extend to all Plasmodium species.

STAR★METHODS

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Yu-Min Chuang (yu-min.chuang@yale.edu).

Materials availability

All stable reagents generated in this study are available from the lead contact with a completed materials transfer agreement.

Data and code availability

All original data generated in this paper will be shared by the lead contact upon request.

This paper does not generate original code.

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

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Mice

4–6 weeks old female Swiss Webster mice and C57BL/6J mice were purchased from Charles River Laboratories. All mice were maintained based on the protocols which were approved by the Yale University Institutional Animal Care & Use Committee (Protocol Permit Number: 2023–07941).

Mosquitoes rearing

A. gambiae were raised at 27°C, 80% humidity, under a 12/12-h light/dark cycle and maintained with 10% sucrose under standard laboratory conditions in the insectary at Yale University. Egg masses were generated via blood meal on naive mice. Uninfected mosquitoes were then housed in a warm (27°C) chamber.

P. berghei infection

P. berghei (NK65 RedStar or ANKA GFP, ATCC) were maintained by serial passage in 6- to 8-week-old female Swiss Webster or C57BL/6 mice. Swiss Webster or C57BL/6 mice were challenged with P. berghei-infected red blood cells by intraperitoneal injection.16 A. gambiae then took a blood meal from the infected mice, when the parasitemia was approximately 1–3%. After blood meals, infected mosquitoes were housed in a cold (21°C) chamber. 17 to 24 days after P. berghei infection, the infected mosquitoes were sorted using the fluorescent signal of the salivary glands. All Plasmodium infections were performed in biosafety level 2 animal facilities, according to Yale University regulations.

METHOD DETAILS

Generation of AgTRIO-KO mosquitoes

gRNA sequences targeting the AgTRIO gene were identified using the CRISPR Design online tool (CHOPCHOP). Three gRNA sequences that had the fewest predicted genomic off-targets were selected (TableS1). Linear double-stranded DNA fragments encoding each gRNA were produced by annealing two partially overlapping oligos to generate AgTRIO gRNA expression plasmids.19 Then, the three double-stranded DNA fragments were individually cloned into a pBluescript vector backbone that contained the U6 snRNA polymerase III promoter (AGAP013557), CRISPR RNA invariable sequences, and the RNA polIII TTTTT terminator. The pKSB-gRNA modules were used to assemble the three gRNA genes into the pDSAR vector via the Golden Gate assembly system (NEB) for embryo microinjection into the A. gambiae docking line X1.28 The backbone plasmid pDSAR with 3xP3-RFP allowed screening of gRNA-positive larvae or mosquitoes by red fluorescence in the eyes.

A QIAGEN Endofree Maxi Kit was used to prepare pDSAR-AgTRIO-gRNA3 and a helper plasmid containing the vasa2:ΦC31 integrase gene (pENTR-R4R3-vasa2-integrase).28,29 The gRNA plasmid (160 ng/μL) and the helper plasmid (200 ng/μL) in a phosphate buffer (0.1 mM NaHPO4 buffer, and 5 mM KCl, pH 6.8) were microinjected into embryos of the docking strain X1.28,30 RFP-positive G0 female mosquitoes were crossed with WT X1 male mosquitoes at a ratio of 1:1. The G1 progeny were examined for RFP-glowing eyes at both the larval and adult stages. Positive G1 mosquitoes were outcrossed with X1 for two generations followed by two generations of self-crossing to enrich the homozygous mosquitoes by the screening of RFP in the larvae before crossing with the vasa-Cas9 strain to generate the AgTRIO knockout mutants.

The gRNA-expressing mosquitoes were crossed with the homozygous Cas9 transgenic strain (vasa2-Cas9; with green fluorescence in the eyes).31 Since the parental gRNA mosquitoes were not fully homozygous, the progeny of this cross were separated into two groups by screening the RFP signal at the larvae stage: the group of larvae with both RFP and GFP signals was gRNA/Cas9 transgenic, and the other group of larvae carrying only the Cas9-GFP transgene serves as the control strain. After blood meal, each female was put in a tube for oviposition. After oviposition, each female adult mosquitoes were selected from the gRNA/Cas9 transgenic group for genomic DNA extraction using the DNEasy Blood and Tissue Kit (69581; QIAGEN). The gRNA-targeted genomic locus was amplified by PCR with flanking primers (TableS1). PCR products were separated with an agarose gel (1%). Only the progeny of the homozygous mutant female was kept, and the selection was continued until all were homozygous mutants. The salivary glands of AgTRIO mutant mosaic mosquitoes were dissected for Western blot analysis to confirm the complete knockout of AgTRIO expression compared with the sibling mosquitoes. The sizes of mosquitoes, including the head, thorax and abdomen, were measured using EVOS microscopy (ThermoFisher).

Western blots

Five salivary glands were put in Laemmli sample buffer (Bio-Rad) and separated by SDS-PAGE using 4–20% Mini-ProteanTGX gels (Bio-Rad). Proteins were transferred onto a 0.45 μm PVDF membrane and then probed with AgTRIO mouse monoclonal antibody (1 ng/ml) or anti-salivary gland extract rabbit antiserum as the primary antibody.16,18 HRP goat anti-rabbit antibody or goat anti-mouse antibody (Invitrogen) was used as the secondary antibody (1:5000) and the images were developed with an LI-COR Odyssey imaging system.

Probing time

5 to 7 days-old adult female mosquitoes, AgTRIO-KO, control or gRNA strain were used for the probing time experiments as described before.9,11 Briefly, 3 mosquitoes were transferred in a 15 mL tube with a 1 × 0.5 cm opening covered with mesh and starved overnight. Mosquitoes were then exposed to an anesthetized Swiss Webster mouse or Hemotek membrane feeder. Probing time is defined as the time taken from the initial insertion of the mouthparts in the skin until the initial engorgement of blood was noted in the abdomen.9,11 If the mosquito required several attempts to intake blood, the probing time of each attempt were added together and the time between attempts was not included. The assay was conducted for 10 min for each mosquito. The group of mosquitoes were de-itemized when interpreted.

Artificial membrane feeding

Mosquitoes were starved for at least 17 h before exposure to blood. Blood was collected from mice infected and treated with EDTA to prevent coagulation. The blood was then transferred into the Hemotek blood reservoir unit. Mosquitoes were then fed at a constant temperature of 37°C using the Hemotek blood feeding system with stretched parafilm as a membrane. For blood feeding, mosquitoes were kept in plastic cups covered with a net and then fed with the blood reservoir unit.

Blood meal amount

Mosquitoes were fed on sedated Swiss Webster mice for 20 min or a Hemotek membrane feeder for 10 min. Unfed mosquitoes were removed after feeding and the whole mosquitoes were frozen immediately. The whole mosquitoes were pulverized in 100 μL distill water to release the blood into the water. Blood-meal volume was then determined by using a colorimetric method.20 Lysate from the above samples were transferred to individual wells of a 96-well plate. The standard curves were generated using the same blood from the mice. The absorbance was measured using a micro plate reader at 540 nm. The same resource blood standard curves were used to calculate engorged mosquito blood-meal volumes.

Vascular leakage

Extravascular leakage after mosquito bites were measured using systemically injected Evans blue dye that binds covalently to serum albumin.32 When there is vascular leakage, the normal endothelial cell barriers are damaged, and albumin can leakage into the extracellular space. Mice were injected intravenously with 50 mg/kg Evans blue (Sigma, #E2129–10G) in PBS and then mosquitoes were allowed to take blood meals on the belly. 1 h after the mosquito bites, the mice were euthanized. Skin samples were acquired by punch biopsy and then placed in 250 μL of formamide. After incubation overnight at 4°C, the skin samples were removed and dye-stained formamide solution was added and analyzed by colorimetric measurement of the dye concentration at 620 nm

Skin cytokine measurement after mosquito bites

3 h after mosquito bites, a punch biopsy was taken at the bite site over the mouse’s ear (3.5 mm, Integra LifeSciences, US). The samples were treated with Dispase II (2.4 U/mL; Roche) in DMEM medium overnight at 4°C and then ground using a grinder homogenizer.33 The lysates were mixed with RIPA lysis buffer (ThermoFisher) with protease inhibitor cocktail (Roche) on ice for 30 min. After centrifugation for 15 min at 12,000× g at 4°C, the lysate supernatant was collected and sent to Eve Technologies for the Mouse Cytokine/Chemokine Array 32-plex (MD-32).

Gene expression and Plasmodium load

The RNeasy Mini Kit (QIAGEN, CA) was used to extract RNA from the salivary glands of the mosquitoes. All extractions followed the manufacturer’s protocols. The iScript RT-qPCR kit (Bio-Rad, CA) was used to generate cDNA from RNA. Using iTaq SYBR Green Supermix (Bio-Rad, CA), real-time PCR was performed on a CFX96 real-time platform (Bio-Rad). PCR involved an initial denaturation at 95°C for 2 min, 50 cycles of 15 s at 95°C, 15 s at 60°C, and 20 s at 72°C. Fluorescence readings were taken at 72°C after each cycle. At the end of each reaction, a melting curve (60°C–95°C) was checked to confirm the identity of the PCR product. The burden of Plasmodium in salivary glands was determined by assessing the expression level of P. berghei 18S rRNA, normalized to A. gambiae actin mRNA. These data were presented as copy numbers of the target gene per 10,000 copies of the housekeeping gene, actin. The primers used for the expression of sporozoite genes are listed in Table S1.

QUANTIFICATION AND STATISTICAL ANALYSIS

GraphPad Prism 10.0 software was used for all statistical analysis and graph generation. Statistical detail for each experiment can be found in the figure legend and results including the number of the biological replicated performed.

For mosquito probing (Figure 2), blood amount intake (Figures 3A and 3B), vascular leakage (Figure 3C), parasite burden in mosquito salivary glands (Figure 4A) and Plasmodium liver burden (Figures 4C and 4D), three or more experiments were performed, using different batches of mosquitoes. Each dots represented individual mosquitoes (Figure 2, 3A–C; 4A) or mice (Figures 4C and 4D). For cytokines responses from the bite sites (Figure 3D), each dot represented a punch biopsy from a single bite site on individual mice ear. Data were analyzed by the Mann-Whitney test to determine the difference and the data were presented as median with the interquartile range (IQR). No data points were excluded. A p-value of <0.05 was considered statistically significant.

Supplementary Material

1

ACKNOWLEDGMENTS

This study was supported by the National Institute of Allergy and Infectious Diseases (RO1 grant AI158615–01, R21 grant AI142708, and R41 grant AI145779 to E.F.) and the Howard Hughes Medical Institute Emerging Pathogens Program.

Figure 1. Generation of AgTRIO KO A. gambiae using CRISPR-Cas9.

(A) Scheme of the gRNA targeting site on AgTRIO. The numbers indicate the positions of the gRNA target regions.

(B) Examples of PCR results of the control (Cntl) and heterozygous and homozygous AgTRIO KO mosquitoes.

(C) Western blots of salivary gland extracts from the gRNA, control and homozygous AgTRIO KO (KO) mosquitoes. Salivary gland extract from five mosquitoes was used per lane. Blots were probed with an AgTRIO monoclonal antibody and anti-salivary gland extract rabbit antiserum.

Figure 2. The absence of AgTRIO reduces the probing efficacy of A. gambiae when taking a blood meal on mice.

(A) The control (n = 22), gRNA strain (n = 32), and AgTRIO KO (n = 22) uninfected mosquitoes fed on the ears of mice for up to 10 min. The time between inserting the mouthpart and a visible blood meal inside the midgut was recorded as the probing time.

(B) Probing attempts were measured as the number of times that mosquitoes inserted their mouthparts into the skin until they successfully obtained a blood meal in the midgut. Samples sizes were as follows: control, n = 16; AgTRIO KO, n = 19; gRNA, n = 32.

(C) The control (n = 38) and AgTRIO KO (n = 38) mosquitoes fed on an artificial membrane feeder for 10 min. The time between the insertion of the mouthpart into the membrane and blood meal inside the midgut was recorded as the probing time. The data are presented as median ± interquartile range (IQR) and pooled from three independent experiments. p < 0.05 using Mann-Whitney U test; NS, not significant.

Figure 3. The absence of AgTRIO changes the amount of blood obtained when feeding on mice but does not affect vascular leakage and cytokine responses at the bite sites.

(A) The control (n = 43), gRNA strain (n = 36), and AgTRIO KO (n = 36) uninfected mosquitoes fed on mice for up to 20 min.

(B) The control (n = 19) and AgTRIO KO (n = 27) mosquitoes fed on an artificial membrane feeder for 10 min. The amount of blood inside mosquitoes was determined by colorimetric assays. The data are presented as median ± IQR and pooled from three independent experiments. p < 0.05 using Mann-Whitney U test.

(C) Vascular leakage after the control (n = 18), gRNA strain (n = 8), or AgTRIO-KO (n = 28) mosquito bite was determined by Evan blue leakage assay. The data are presented as median ± IQR and pooled from three independent experiments. p < 0.05 using Mann-Whitney U test.

(D) The control (n = 5) or AgTRIO KO uninfected mosquitoes (n = 6) were allowed to take a blood meal on the ears of mice. After 3 h, the bite site was removed by punch biopsy, and the level of cytokines was determined by immunobead assay. Median ± IQR using Mann-Whitney U test.

Figure 4. Plasmodium transmission to mice is increased in AgTRIO KO A. gambiae.

(A) The salivary glands of the control or AgTRIO KO mosquitoes were collected 15 days (n = 23 and 25) or 21 days (n = 19 and 28) after taking blood meals on P. berghei-infected mice. The Plasmodium burden in salivary glands was determined by the expression of P. berghei 18S by RT-PCR after being normalized to the expression of mosquito actin.

(B) The sporozoites were collected from the salivary glands 17 days after mosquitoes took blood meals from P. berghei-infected mice. 300 sporozoites from the control or AgTRIO-KO mosquitoes were injected intradermally into the left ears of individual C57BL/6 mice. After 40 h, the livers were dissected, and the Plasmodium infection was determined by RT-PCR. Sample size: n = 13 from control mosquitoes and n = 14 from AgTRIO KO mosquitoes.

(C) C57BL/6 mice were exposed to 3 P berghei-infected control or AgTRIO KO mosquitoes. 40 h later, livers were dissected, and the Plasmodium infection level was determined by RT-PCR. Sample size: n = 14 mice from control mosquitoes and n = 15 mice from AgTRIO-KO mosquitoes. The data are presented as median ± IQR and were pooled from three independent experiments. p < 0.05 using Mann-Whitney U test.

KEY RESOURCES TABLE REAGENT or RESOURCE	SOURCE	IDENTIFIER	
	
Antibodies	
	
HRP goat anti-rabbit antibody	Invitrogen	Cat #: 31460; RRID: AB_228341	
HRP goat anti-mouse antibody	Invitrogen	Cat #: 31430; RRID: AB_228307	
	
Chemicals, peptides, and recombinant proteins	
	
Evans blue	Sigma	E2129-10G	
Formamide	Sigma	551384	
RIPA lysis buffer	ThermoFisher	89900	
Protease inhibitor cocktail	Roche	11836153001	
TRIzol	Thermo	15596018	
RNeasy Mini Kit (250)	QIAGEN	Cat No./ID: 74106	
	
Critical commercial assays	
	
Golden Gate assembly system	New England biolabs	R0739S	
Endofree plasmid maxi kit	QIAGEN	12362	
DNEasy Blood and Tissue Kit	QIAGEN	69581	
	
Experimental models: Organisms/strains	
	
A. gambiae Cas9 strain	Dong et al.19	N/A	
A. gambiae gRNA strain	This lab	N/A	
A. gambiae AgTRIO-KO strain	This lab	N/A	
Plasmodium berghei (ANKA GFPcon	ATCC	MRA-865	
259cL2)			
Plasmodium berghei NK65 RedStar	ATCC	MRA-905	
Swiss Webster mice	Charles River Lab	Cat# CRL:24	
C57BL/6J mice	Charles River Lab	Cat# CRL:27	
	
Oligonucleotides	
	
See Table S1	This study	N/A	
	
Recombinant DNA	
	
pDSAR-AgTRIO-gRNA3	This study	N/A	
pENTR-R4R3-vasa2-integrase	Volohonsky et al.28	Addgene: 62299	
	
Software and algorithms	
	
CHOPCHOP	University of Bergen, Bergen, Norway	https://chopchop.cbu.uib.no/	
Graphpad Prism	La Jolla, CA	Version 10	

Highlights

AgTRIO knockout mosquitoes show a diminished host probing capacity

AgTRIO knockout mosquitoes require repetitive probing to complete a blood meal

Increased probing results in enhanced Plasmodium transmission to the vertebrate host

DECLARATION OF INTERESTS

The authors declare no competing interests.

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2024.114600.
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