Cardinium Localization During Its Parasitoid Wasp Host's Development Provides Insights Into Cytoplasmic Incompatibility

Abstract

Arthropods harbor heritable intracellular symbionts that may manipulate host reproduction to favor symbiont transmission. In cytoplasmic incompatibility (CI), the symbiont sabotages the reproduction of infected males such that high levels of offspring mortality result when they mate with uninfected females. In crosses with infected males and infected females, however (the "rescue" cross), normal numbers of offspring are produced. A common CI-inducing symbiont, Cardinium hertigii, causes variable levels of CI mortality in the parasitoid wasp, Encarsia suzannae. Previous work correlated CI-induced mortality with male development time in this system, although the timing of Cardinium CI-induction and the relationship between development time and CI mortality was not well understood. Here, using a combination of crosses, manipulation of development time, and fluorescence microscopy, we identify the localization and the timing of the CI-induction step in the Cardinium-E. suzannae system. Antibiotic treatment of adult Cardinium-infected males did not reduce the mortality associated with the CI phenotype, suggesting that CI-alteration occurs prior to adulthood. Our results suggest that the alteration step occurs during the pupal period, and is limited by the duration of pupal development: 1) Encarsia produces most sperm prior to adulthood, 2) FISH localization of Cardinium in testes showed an association with sperm nuclei throughout spermatogenesis but not with mature sperm, and 3) two methods of prolonging the pupal period (cool temperatures and the juvenile hormone analog methoprene) both caused greater CI mortality, suggesting the degree of alteration is limited by the duration of the pupal stage. Based on these results, we compare two models for potential mechanisms of Cardinium sperm modification in the context of what is known about analogous mechanisms of Wolbachia, a more extensively studied CI-inducing symbiont.

Keywords: cardinium; cytoplasmic incompatibility; encarsia; parasitoid; spermatogenesis; symbiosis; wolbachia.

FIGURE 1

Effects of adult antibiotic (rifampicin) exposure on (A) relative density of Cardinium in male wasps determined by qPCR (Cardinium gyrB copy number/host ef1A copy number), and (B) resulting CI-induced offspring mortality. Sample size in A = 8 samples with three technical replicates. Sample sizes for (B) are given above or below whisker plots. Statistical comparisons between honey-fed control and rifampicin-treated adults were determined by Mann-Whitney U-test in (A) and, in (B), by quasibinomial logistic regression between equivalent cross types in rifampicin and control treatments. Asterisks indicate statistical significance. ***p < 0.001.

FIGURE 2

Cardinium localization in “white” pupal testes. The distal portion of the testis is at the top of all panels. (A) White E. suzannae male pupa. (B) Entire pupal testis showing DAPI-stained host nuclei (blue) and (C) also showing cy3-stained Cardinium 16S rRNA (green). (D) Magnified distal region, showing spermatocytes and (E) Cardinium associations with host nuclei in the same region. The circled region shows a cyst with condensed nuclei beginning to align in the cup formation representing entry into spermiogenesis. (F) Magnified mid-region of testis, showing interphase spermatocyte nuclei and (G) Cardinium relative to host nuclei. The arrowhead in F and G refers to an interphase nucleus engulfed by Cardinium cells and the circled region shows nuclei associated with a dense patch of Cardinium cells. (H) Magnified apical region of testis. (I) Location of Cardinium in apical region of testis, which contains fewer Cardinium cells relative to the rest of the testis.

FIGURE 3

Cardinium localization in “red-eyed” pupal testes. (A) Red-eyed E. suzannae male pupa. (B) Entire red-eye pupal testis oriented from top (distal) – down (apical) showing DAPI-stained host nuclei (blue) and (C) cy3-stained Cardinium 16S rRNA (green) with host nuclei. (D) Magnified distal region of testis, with DAPI-stained nuclei and (E) cy3-stained Cardinium 16S rRNA (green) with host nuclei. Solid circles show a cyst with elongating nuclei forming the characteristic c-shape. What appears to be empty space in these cysts is occupied by the cytoplasm of the elongating sperm tail. (F) Magnified mid-region of testis showing DAPI-stained host nuclei (blue) and (G) cy3-stained Cardinium 16S rRNA (green) with host nuclei. Here the Cardinium cells appear more evenly distributed across cysts than in the white pupal stage. Arrowheads show representative individual nuclei that have not begun elongating with closely associating Cardinium cells.

FIGURE 4

Cardinium localization in “black” pupal testes. (A) Black E. suzannae male pupa. (B) Entire black pupal testis oriented from lower left (apical) to upper right (distal) showing DAPI-stained host nuclei (blue) and (C) cy3-stained Cardinium 16S rRNA (green) with host nuclei. (D) Magnified mid-region showing DAPI-stained host nuclei (blue) and (E) cy3-stained Cardinium 16S rRNA (green) with host nuclei. Circled regions show cysts with condensed nuclei forming the cup formation indicative of entry into spermiogenesis. Arrowheads show individual interphase nuclei with closely associating Cardinium. (F) Magnified distal region of testis with DAPI-stained nuclei and (G) cy3-stained Cardinium 16S rRNA (green) with host nuclei. The circled region in (G) shows a cyst with elongating nuclei that appears to be losing its resident Cardinium. Empty space in these cysts is occupied by the cytoplasm of the elongating sperm tail. Arrowheads show the larger nucleus of either a somatic cyst cell or a trophocyte (nutritional cells). (H) Magnified view of the seminal vesicle containing mature sperm. No Cardinium cells are evident.

FIGURE 5

Cardinium localization in adult testes two days post-emergence. (A) Adult male E. suzannae. (B) Entire adult testis oriented from top (distal) to bottom (apical) with DAPI-stained host nuclei only (blue) and (C) DAPI + cy3-stained Cardinium cells (green). (D) Magnified distal region of testis with DAPI-stained nuclei and (E) cy3-stained Cardinium with host nuclei. Circled region shows a cyst in spermiogenesis with elongating nuclei. Empty space in these cysts is occupied by the cytoplasm of the elongating sperm tail. (F) Magnified mid-region showing DAPI-stained host nuclei and (G) cy3-stained Cardinium with host nuclei. Arrowheads show the larger nucleus of a somatic cell.

FIGURE 6

Effects of cold temperature and methoprene on Cardinium-induced CI and E. suzannae male development (A) Effects of early or late male pupal exposure to cool temperatures (20°C day/17°C night) on Cardinium-induced CI mortality of offspring. The early pupal treatment lasted from the onset of pupation, through the white and red-eyed stages until the onset of cuticular melanization. The late pupal treatment began at the onset of cuticular melanization, through the black pupal stage, and ended at adult emergence. (B) Effects of male wasp exposure to methoprene and cool temperatures (20°C day/17°C night) on duration of pupal development and (C) CI-induced offspring mortality. The numbers above or below the box and whisker plots represent biological replicates (n = 10–48 for all treatments). Rescue and CI crosses are denoted by R and CI, respectively, except in (A), which only shows CI crosses. In (A) and (C), logistic regression with a quasibinomial distribution was used to compare offspring mortality of the control R or CI cross with each of the comparable treatment crosses (or to the control CI cross in A). In (B), significant differences from the control treatment were determined by multiple pairwise Student’s T-tests with Benjamini-Hochberg corrected p-values. Asterisks denote P values (*p = 0.05, **p < 0.01, and ***p < 0.001).

FIGURE 7

Models for Cardinium sperm alteration. (A) Pre-fertilization “host modification” model, in which Cardinium produces a CI effector during early spermatogenesis stages when the symbiont is close to spermatocyte nuclei. The host target, likely a protein involved in spermiogenesis-associated nuclear reorganization, becomes available for alteration during elongation or spermiogenesis. CI-altered mature sperm enter seminal vesicle. (B) Post-fertilization “toxin-antidote” model, in which Cardinium stockpiles sperm cells with the CI effector throughout spermatogenesis until symbiont removal during spermiogenesis. The CI effector is packaged into spermatids, but the final host target, potentially a protein involved in re-modeling of sperm chromatin from its elongated state, is available in the egg cytoplasm. Mature sperm must be enriched for the CI effector at some threshold to cause cellular defects in the egg and mortality.