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. 2007 Feb 13;104(7):2121-6.
doi: 10.1073/pnas.0608407104. Epub 2007 Feb 6.

Reactive oxygen species detoxification by catalase is a major determinant of fecundity in the mosquito Anopheles gambiae

Randall J DeJong  1 , Lisa M Miller, Alvaro Molina-Cruz, Lalita Gupta, Sanjeev Kumar, Carolina Barillas-Mury
Affiliations

Reactive oxygen species detoxification by catalase is a major determinant of fecundity in the mosquito Anopheles gambiae

Randall J DeJong et al. Proc Natl Acad Sci U S A. .

Abstract

The mosquito Anopheles gambiae is a primary vector of Plasmodium parasites in Africa. The effect of aging on reproductive output in A. gambiae females from three strains that differ in their ability to melanize Plasmodium and in their systemic levels of hydrogen peroxide (H2O2), a reactive oxygen species (ROS), was analyzed. The number of eggs oviposited after the first blood meal decreases with age in all strains; however, this decline was much more pronounced in the G3 (unselected) and R (refractory to Plasmodium infection) strains than in the S (highly susceptible to Plasmodium) strain. Reduction of ROS levels in G3 and R females by administration of antioxidants reversed this age-related decline in fecundity. The S and G3 strains were fixed for two functionally different catalase alleles that differ at the second amino acid position (Ser2Trp). Biochemical analysis of recombinant proteins revealed that the Trp isoform has lower specific activity and higher Km than the Ser isoform, indicating that the former is a less efficient enzyme. The Trp-for-Ser substitution appears to destabilize the functional tetrameric form of the enzyme. Both alleles are present in the R strain, and Ser/Ser females had significantly higher fecundity than Trp/Trp females. Finally, a systemic reduction in catalase activity by dsRNA-mediated knockdown significantly reduced the reproductive output of mosquito females, indicating that catalase plays a central role in protecting the oocyte and early embryo from ROS damage.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Age-associated loss of fecundity in the G3 and R strains. (A) Average number of eggs per female oviposited ± SE. (B) Average number of larvae produced per female ± SE. The first blood meal was given at 4, 6, or 8 days PE (n = three groups of 10 females each). (C) Effect of supplemental antioxidant feeding (vitamin C or uric acid) on fecundity when females were blood fed at 8 days PE. Both antioxidant treatments restored fecundity of G3 and R to levels not significantly different from S females. The same antioxidant treatment was deleterious to S females; all of them died within 48 h after blood feeding (indicated by †). Asterisks indicate values significantly lower than all others (P < 0.003, ANOVA).
Fig. 2.
Fig. 2.
Catalase mRNA and protein expression over time in developing oocytes. (A) Relative amounts of catalase mRNA in ovaries at times PBM as measured by quantitative PCR normalizing with ribosomal protein S7. (B) Enzymatic activity of catalase in ovary homogenates from G3 females normalized for total protein in the extract. (C–G) Catalase-mediated DAB staining of whole oocytes. (Scale bars: in C, 30 μm; in D–G, 150 μm.) (C) Developing oocytes (24 h PBM) are positive for DAB staining (brown), whereas unstained oocytes are clear (not shown). (D–G) Mature oocytes (72 h PBM) accumulate catalase protein. (D) Unstained oocyte. (E) Oocyte after DAB staining (dark brown). (F) DAB staining is minimal in oocytes incubated with AT, a catalase inhibitor. (G) AT is removed by extensive washing with PBS, and DAB staining is restored, indicating that AT inhibition is reversible.
Fig. 3.
Fig. 3.
Enzymatic activities of Ser and Trp catalase alleles. (A) Effect of pH on catalase enzymatic activity in ovary homogenates from S (Ser allele) and G3 (Trp allele) females, normalized for total protein in the extract. Means are plotted ± SE. Ser allele, filled circles; Trp allele, open circles. (B) Effect of pH on enzymatic activity of purified recombinant Ser and Trp catalase isoforms. (C) Effect of temperature on enzymatic activity of purified recombinant Ser and Trp catalase isoforms. (D) Effect of substrate concentration on initial velocity of catalysis and determination of Km and Vmax of purified recombinant Ser and Trp catalase isoforms. (E) Native gel electrophoresis of recombinant Ser and Trp isoforms, showing that the Trp isoform has relatively less protein in tetramer form. (F) SDS/PAGE of Ser and Trp recombinant protein isoforms shows a single band.
Fig. 4.
Fig. 4.
Effect of reduced catalase activity on fecundity. (A) Association between catalase allele genotypes and fecundity in R females. Asterisk indicates that Trp/Trp individuals produced significantly less offspring than those homozygous for the Ser allele. The table below the graph indicates the percentage of mosquitoes that exhibited oviposition failure (Ov. f.), which was significantly different among the groups (χ2 test, P = 0.005). (B and C) Effect of catalase knockdown on fecundity. (B) Relative amounts of catalase mRNA in G3 mosquitoes after injection with catalase dsRNA (dsCAT) compared with controls injected with LacZ dsRNA (dsLacZ). A 65% knockdown in abdomen catalase mRNA levels was achieved. (C) Mean number of larvae produced per female (± SE) decreased by 45% in dsCAT-knockdown vs. control G3 mosquitoes (t test, P = 0.046).
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