Fig 1.
Expression, quantification and detection of mJHBP in Ae. aegypti.
A. Detection of mJHBP protein in developmental stages by SDS-PAGE (NuPage) and Western blotting of whole mosquito homogenates (protein standard sizes in kilodaltons are shown on left). E1: Embryo (n = 20), E2: Egg (n = 20), L1: 1st instar larvae (n = 20), L2: 2nd instar larvae (n = 20), L3: 3rd instar larvae (n = 20), L4: 4th instar larvae (n = 20), P: pupae (n = 5), F: adult female (n = 5), M: adult male (n = 5). Samples were reduced with NuPAGE sample reducing agent (stabilized dithiothreitol) prior to electrophoresis. Molecular size standards are shown to the left of the gel. B. Quantitation of Ae. aegypti mJHBP protein in individual pupae (P) and adult female mosquitoes (0, 24, 48 and 72 h post emergence) measured by ELISA. Each bar represents the mean (ng / individual ± SEM) quantity of mJHBP from 15 mosquitoes (3 replicates of 5 insects in each experiment). There are no statistical differences between timepoints or developmental stages. C. Visualization of Ae. aegypti mJHBP protein in the fat body (FB) by confocal microscopy. mJHBP, green; actin, magenta. D. Visualization of Ae. aegypti mJHBP protein in pericardial cells (asterisks, DV = dorsal vessel) by confocal microscopy. mJHBP, green; actin, magenta; Scale bar, 30 μm.
Fig 2.
Effect of mJHBP deficiency on developmental and reproductive aspects in Wild Type (WT) and mJHBP-/- (KO) lines.
A. Mean time to pupation (days) from egg hatching (day 0) to the onset of pupation for WT and KO mosquitoes (4 biological replicates of 20 larvae). B. Mean duration of the pupal stage (days) for WT and KO mosquitoes (4 biological replicates of 20 pupae). C. Mean wing lengths (±SEM) for WT (n = 30) and KO (3 biological replicates of 10 adults) female mosquitoes. D. Mean number of eggs (±SEM) laid by WT and KO females (3 biological replicates of 9–10 adults). E. Quantification of triglyceride levels in WT and KO in newly emerged adult females (6 h, 12 h, 24 h, 48 h, 72 h and 96 h post adult emergence) (2 biological replicates of 2 mosquitoes for each time point). F. Mean JH III levels (±SE) in WT and KO mosquito hemolymph quantified by mass spectrometry (5 biological replicates, of 5 mosquitoes). None of these characteristics differed significantly (p ≤0.05) between WT and KO lines (Mann-Whitney U test).
Fig 3.
Effect of mJHBP deficiency on innate immune responses after bacterial challenge.
A. mJHBP-/- (KO) mosquitoes are unable to control bacterial infection. Bacterial counts (CFU) from 3 biological replicates of 2–4 individual WT and KO mosquitoes infected with E. coli (OD600 = 0.1) 24 (n = 8), 48 (n = 10) and 72 h (n = 11) post-infection. Significance levels (Mann-Whitney U test) are indicated as asterisks (**** p ≤ 0.0001, *** ≤ 0.001, ** ≤ 0.01, * ≤ 0.05). Median values are shown as horizontal lines. Mortality of E. coli-injected mosquitoes was negligible. B. Effect of mJHBP deficiency on the survival of mJHBP-/- (KO) mosquitoes. Kaplan-Meier survival curves for WT (n = 20) and KO (n = 17, two biological replicates) females infected by S. marcescens (OD600 = 0.05) shows that KO mosquitoes succumbed earlier than WT females. The median survival between WT and KO mosquitoes was statistically significant (Log-rank test, p≤0.0001). C,D. Effect of mJHBP deficiency on the expression of the antimicrobial peptides defensin A (C) and cecropin A (D) in the fat body of E. coli-challenged females measured at 6, 24, 48 and 72 h post-infection (n = 9, 3 biological replicates of 3 individuals). mJHBP-/- (KO) mosquitoes exhibited a 24 h delay in the induction of both AMPs. Changes in expression were calculated by comparing the ΔCt of infected mosquitoes with that of LB-injected mosquitoes (2-ΔΔCt). Black arrow: time of induction to maximum expression level in the WT strain = 24 h; Magenta arrow: time of induction to maximum expression in the KO strain = 48 h. Data were analyzed using the Mann-Whitney U test (**** p ≤ 0.0001). Again, mortality of E. coli-infected mosquitoes was negligible.
Fig 4.
Effect of mJHBP deficiency on the expression of markers of immune function in hemocytes, fat body and midgut tissues.
Relative expression levels for defensin A (A) and cecropin A (B) in hemocytes from E. coli-challenged WT and KO females measured at 24 and 48 h post-infection (n = 6, 3 biological replicates of 2 individuals). Relative expression levels for nitric oxide synthase (NOS) in fat body (C) and hemocytes (D) from E. coli- challenged females measured at 24 and 48 h post-infection (n = 6, 3 biological replicates of two individuals). Relative expression levels for defensin A (E), cecropin A (F), attacin B (G), and gambicin (H) in midguts from E. coli-challenged WT and KO females measured at 24 and 48 h post-infection (n = 6, 3 biological replicates of 2 individuals). Data were analyzed using the Mann-Whitney U test (** p ≤ 0.01).
Fig 5.
Mutagenesis and ligand binding profiling of mJHBP.
A. The JH III binding site is located in the N-terminal domain (N-term). In the wild type mJHBP-JH III complex helix α-13 of the C-terminal domain covers the ligand access channel (red). The α-13 position in the presumed open form of the ligand-free protein would be folded back over the C-terminal domain (blue). In the ΔCt mutant α-13 is missing, apparently destabilizing the mJHBP-JH III complex. B. Binding site detail showing hydrogen bonding between the Tyr129 hydroxyl group and the JH III epoxy group. This residue is mutated to phenylalanine in the Y129F and VFYF mutants. In the VFYF mutant Val68 is also mutated to phenylalanine (magenta), further occluding the binding pocket. C. Three site-directed mutants of mJHBP were evaluated for interaction with JH III using isothermal titration calorimetry (ITC). Traces showing heats of interaction reveal highly enthalpic binding of JH III with the WT protein. Injection heats decrease with increasing modification of the protein (WT > Y129F > VFYF > ΔCt).
Fig 6.
Effect of wild type and mutant mJHBP protein injection on bacterial replication and induction of antimicrobial peptides in fat bodies and hemocytes from E. coli-challenged females.
Data shown in A-C compares the activity of wild type mJHBP protein and three JH-binding deficient mutants. A. The ability of mJHBP site-directed mutants to rescue the ability of mJHBP-/- (KO) mosquitoes to control bacterial infection correlates with JH binding capability measured in ITC experiments. The order of immunomodulatory potency for the proteins is: WT > Y129F > VFYF > ΔCt. The responses of infected WT females are shown for comparison. Statistical comparisons are shown between buffer-injected females and females injected with WT mJHBP or its mutants. Significance levels (Mann-Whitney U test) are indicated with asterisks (**** p ≤ 0.0001, *** ≤ 0.001, ** ≤ 0.01). Sample sizes: WT (n = 13, 4 biological replicates), WT+mJHBP (n = 6, 3 replicates), WT+ΔCt (n = 6, 3 replicates), KO (n = 11, 3 replicates), KO+mJHBP (n = 12, 4 replicates), KO+ΔCt (n = 8, 3 replicates), KO+Y129F (n = 15, 5 replicates), KO+YFVF (n = 12, 4 replicates). Median values are shown as horizontal lines. The injection of biologically active mJHBP into mJHBP-/- (KO) mosquitoes rescues the early induction of the AMPs defensin A (B) and cecropin A (C) in the fat body at 24 h after bacterial infection. AMP expression in protein-injected, E. coli-infected mosquitoes was determined relative to protein-injected, HBSS-injected (no infection) individuals by quantitative PCR using the 2-ΔΔCt method. Site-directed mutant proteins vary in effectiveness in restoring AMP production corresponding to their JH III binding affinity (WT > Y129F > VFYF > ΔCt). The response of infected WT females is shown for comparison. Sample sizes: WT (n = 6, 3 biological replicates), KO (n = 6, 3 replicates), KO+mJHBP (n = 7, 3 replicates), KO+ΔCt (n = 6, 3 replicates), KO+Y129F (n = 6, 3 replicates), KO+VFYF (n = 6, 3 replicates). Treatments were compared using the Mann-Whitney U test (** p ≤ 0.01, * ≤ 0.05). The injection of biologically active mJHBP into KO mosquitoes rescues the early induction of the antimicrobial peptides defensin A (D) and cecropin A (E) in hemocytes at 24 h after bacterial infection. Sample sizes: (n = 6, 3 biological replicates). Data were analyzed using the Mann-Whitney U test (** p ≤ 0.01).
Fig 7.
mJHBP-deficient mosquitoes exhibit impaired hemocyte development (lower hemocyte counts) and increased granulocyte differentiation.
Relative abundance (Mean number ± SEM) of total hemocytes (A), prohemocytes (B) and granulocytes (C) per female in newly-emerged WT and KO lines. Relative proportion (%) of prohemocyte (D) and granulocyte (E) subpopulations in in newly emerged WT and KO lines. Relative abundance (Mean number ± SEM) of total hemocytes (F), as well as the proportions of prohemocytes (G), and granulocytes (H) perfused from newly emerged WT and KO adults reared to eclosion after injection of fourth instar larvae with buffer or biologically active mJHBP. Significance levels (Mann-Whitney U) are indicated with asterisks (**** p ≤ 0.0001, *** ≤ 0.001, ** ≤ 0.01, * ≤ 0.05). In panels A-E two biological replicates of 10 female mosquitoes each were counted. In panels F-H, two biological replicates of 4–7 mosquitoes each were performed.
Fig 8.
Phagocytic capabilities of hemocytes perfused from newly emerged WT and KO females.
Phagocytic capacity of hemocyte from WT (A) and KO (B) lines. Data shown represents the frequency distributions of fluorescent pHrodo E. coli bioparticles per individual granulocyte (phagocyte). Two biological replicates were performed in which 10 granulocytes per mosquito from at least three fields were counted. In each replicate 5 mosquitoes were analyzed. C. Effect of biologically active and mutant mJHBP protein injection on the phagocytic activity (mean % ±SEM) of granulocytes in WT and KO lines. Two biological replicates were performed in which granulocytes were counted as in panels A and B. In each replicate, granulocytes from 7–12 mosquitoes were evaluated. Significance levels (Mann-Whitney U test) are indicated with asterisks (**** p ≤ 0.0001, * ≤ 0.05).
Fig 9.
Effect of mJHBP deficiency on the expression of Toll, IMD, and JAK/STAT pathway components in response to bacterial challenge.
Gene expression comparison between WT and KO females for: A. IMD (IMD Pathway); B. STAT (JAK/STAT pathway), C. MyD88 (Toll pathway), D. Rel1 (Toll pathway), E. Rel2 (IMD pathway), F. Cactus (Toll pathway), G. Caspar (IMD pathway), H. PGRP-S1. Fat bodies of infected WT and KO mosquitoes were dissected at 6, 24, 48, and 72 h post-infection. Each time point represents 50 mosquitoes (5 biological replicates of 10 mosquitoes each). Significance levels (Mann-Whitney) are indicated with asterisks (p ** ≤ 0.01).