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Sci Rep
2017 Jun 12;71:3214. doi: 10.1038/s41598-017-03157-2.
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Functional characterization of Aquaporin-like genes in the human bed bug Cimex lectularius.
Tsujimoto H
,
Sakamoto JM
,
Rasgon JL
.
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The bed bug Cimex lectularius is a blood-feeding re-emerging annoyance pest insect that has the ability to transmit Trypanosoma cruzi under experimental laboratory conditions. Aquaporins (AQPs) are water channel proteins that are essential in biological organisms. C. lectularius are constantly exposed to water-related stress, suggesting that AQPs may offer novel control avenues. We identified and cloned four AQPs from C. lectularius, assessed tissue and lifestage-specific expression, and characterized biochemical functions in vitro and in vivo. We identified an efficient water-specific AQP (ClAQP1), two aquaglyceroporins (ClGlp1 and ClGlp2) and a homolog of Drosophila melanogaster big brain (ClBib). ClGlp1 was only functional when co-expressed with the water-specific AQP. Simultaneous RNAi gene silencing of ClAQP1 and ClGlp1 significantly reduced water and urea excretion post blood feeding. The Bib homologue was enriched in embryos, exclusively expressed in ovaries, and when silenced, dramatically increased bug fecundity. Our data demonstrate that AQPs have critical roles in excretion, water homeostasis and reproduction in C. lectularius, and could be potential targets for control in this notorious pest.
Figure 1. Maximum likelihood phylogenetic tree of identified C. lectularius AQP-like gene sequences (Red). Numbers at notes represent percent bootstrap values. Taxa accession numbers are listed in Table S1.
Figure 2. Lifestage and tissue-specific expression of C. lectularius AQP-like genes quantified by quantitative real-time PCR. (A) Lifestage-specific expression. (B) tissue-specific expression in adult female bugs. ClAQP1 and ClGlp1 have almost superimposable expression patterns, with primary expression in the gut and malpigian tubules. ClGlp2 is primarily expressed in the carcass. ClBib is only expressed in the ovaries. SG = salivary glands, Mg = midgut, MT = malpigian tubules, OV = ovaries, Sp = spermalege, C = carcass. The graph shows mean ± SD.
Figure 3. Water and solute transport activity of C. lectularius AQP genes expressed individually in Xenopus oocytes. (A) ClAQP1 transports water, and is inhibited by mercury. ClGlp1 and ClBib do not transport water. ClClp2 moderately transports water. (B) Water transport ability of ClAQP1 is not altered by co-expression with ClGlps. (C) ClGlp1 does not transport glycerol when expressed by itself, but showed high efficiency glycerol transporter activity when co-expressed with ClAQP1. Glycerol transport ability of ClGlp2 is enhanced when co-expressed with ClAQP1. (D) ClGlp1 does not transport urea when expressed by itself, but showed high efficiency urea transporter activity when co-expressed with ClAQP1. Urea transport ability of ClGlp2 is enhanced when co-expressed with ClAQP1. The graph shows mean ± SEM. Asterisks in the bars represent statistical significance (P < 0.001) in comparison to control; asterisks above the bars represent statistical significance (P < 0.001) comparing between the groups below the horizontal line.
Figure 4. RNA interference (RNAi) is highly efficient in C. lectularius. Controls were injected with dsRNA against GFP. (A) RNAi of ClAQP1. (B) RNAi of ClGlp1. (C) Simultaneous knockdown of ClAQP1 and ClGlp1. DKD = double knockdown. The graph shows mean ± SD.
Figure 5. Lack of off-target effect of dsRNA injection. (A) ClGlp1 expression in dsClAQP1 (or control dsGFP) injected bed bugs. (B) ClAQP1 expression in dsClGlp1 (or dsGFP) injected bed bugs. (C) ClGlp2 expression in dsClAQP1/dsGlp1 (or dsGFP) injected bed bugs. Dpi: days post injection. The graph shows mean ± SD.
Figure 6. Effect of RNAi on bug excretion after a bloodmeal, as measured by weight loss. All controls were injected with dsRNA against GFP. (A) RNAi of ClAQP1. (B) RNAi of ClGlp1. (C) Double knockdown of ClAQP1 and ClGlp1. The graph shows mean ± SEM.
Figure 7. Effect of RNAi on urea excretion after a bloodmeal. All controls were injected with dsRNA against GFP. (A) RNAi of ClAQP1. (B) RNAi of ClGlp1. (C) Double knockdown of ClAQP1 and ClGlp1. The graph shows mean ± SEM.
Figure 8. Effect of RNAi against ClBib on offspring production. Bugs with reduced ClBib expression oviposited significantly more eggs compared to GFP dsRNA-injected controls. The graph shows mean ± SEM.
Figure 9. Pull-down assay to examine interaction between ClAQP1 and ClGlp1: X. laevis oocytes expressing His-ClAQP1 and myc-ClGlp1 or myc-ClGlp1 alone (control) were used to extract proteins, which were subjected to pull-down assay using Ni2+ NTA agarose matrix (Invitrogen). Presence of myc-ClGlp1 in the eluent was assessed by Western blot. Crude protein extracts were loaded as control.
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