Characterizing oogenesis and programmed cell death in the eastern tree hole mosquito Aedes (Protomacleaya) triseriatus

Abstract

Oogenesis in flies manifests as a carefully orchestrated cascade of developmental gates and growth events, punctuated by programmed cell death (PCD) and follicular resorption events. In anautogenous mosquitoes, a blood meal stimulates growth of primary follicles, but the timing of developmental stages is species-specific, and few species have been characterized. Here, we characterize the first gonotrophic cycle of oogenesis in Aedes triseriatus (Diptera: Culicidae), the principal vector of La Crosse Virus (LACV), a major cause of pediatric encephalitis in North America. We note significant differences in the timing and appearance of developmental stages from previous studies of other mosquito species, particularly Aedes aegypti. We also describe the appearance and timing of PCD events including atresia, nurse cell death, and follicular epithelium death and show that the majority of follicular epithelium cells do not undergo apoptosis during oogenesis but persist in the ovariole at least until the second gonotrophic cycle. This thorough characterization of oogenesis and PCD in Ae. triseriatus, through which LACV must persist in order to achieve filial infection, also serves as a baseline to study host-pathogen interactions during transovarial transmission.

Keywords: apoptosis; atresia; autophagy; nurse cell death; ovarian development.

Figure 1

Follicle morphology post bloodmeal. Live NR stained primary follicles from 3-day old Ae. triseriatus during (A) early initiation phase IIIa at 8 hpbm, (B) late initiation phase IIIa at 12 hpbm, (C) trophic phase IIIb at 24 hpbm, (D) trophic phase IVa at 46 hpbm, (E) trophic phase IVb at 55 hpbm and (F) 60 hpbm, (G) post trophic phase V at 72 hpbm and (H) 96 hpbm. Scale bars = 100 µM and 500 µM. Secondary and tertiary follicles within the ovariole visible in all parts except (E) For an illustration of follicle morphology please see Figure S1 .

Figure 2

Quantifying changes in follicle morphology. The size and shape of follicles including the oocyte and nurse cell compartments were measured over time following a bloodmeal. (A) Follicular area over time n = 30, (B) follicular length, width and ratio (Length : Width) over time n = 50, (C) comparison of oocyte and nurse cell area as a percentage of inner follicle area over time n = 15. Data are the average of 3 or more biological replicates (± SEM).

Figure 3

Characterizing follicular atresia. (A) Representative NR stained healthy follicles, atretic follicles (asterisks), and unstained secondary follicles (arrowheads) at 24 hpbm. Scale bar = 100 µM. (B) The proportion of atretic follicles per ovary over time post bloodmeal. (C) Length of healthy vs atretic follicles over time. Data are the average of 3 replicates (± SEM).

Figure 4

Follicular atresia is apoptotic in Ae. triseriatus. (A) Early-stage atresia in a primary follicle with intact DAPI positive nurse cell nuclei and apoptotic TUNEL positive follicular epithelial cells. (B) An atretic and normally developing primary follicle. Advanced atresia with TUNEL positive follicular epithelia is evident in the atretic primary follicle (arrowhead); note no clear definition between follicular epithelium, oocyte, and nurse cells. As compared to a cross section of a normally developing primary follicle above. (C) Positive control primary and secondary follicles treated with DNase I showing TUNEL positive nuclei in follicular epithelial cells, nurse cells, and the oocyte (asterisk). Nuclei (DAPI), actin (Phalloidin), and TUNEL staining channels shown. Scale bar = 100 µM. For an illustration of follicle morphology please see Figure S1 .

Figure 5

Timing Nurse Cell Death (NCD). (A) NR stained NCD positive follicles at 60 hpbm with 1 or more dying nurse cells (arrowheads). (B) Proportion of NCD positive follicles per ovary over time. (C–E) Acridine orange stained nurse cells at 58 hpbm displaying (C) impermeable nurse cells pre-NCD, (D) NCD positive nurse cells with intact nuclei, and (E) NCD positive nurse cells with condensed and degraded nuclei. Scale bars = 100 µM. Data are the average of 3 or more biological replicates (± SEM).

Figure 6

Apoptotic cell death in nurse cells and follicular epithelium is asynchronous. (A) Healthy primary follicles at 60 hpbm during mid-oogenesis displaying various stages of NCD and sporadic follicular epithelial death. (B) Healthy primary follicle during late-oogenesis as 96 hpbm undergoing follicular epithelial sloughing with majority of follicular epithelial nuclei intact. (C) DNase I positive TUNEL staining control of healthy primary follicle with follicular epithelia during late-oogenesis at 96 hpbm. Nuclei (DAPI), actin (Phalloidin), and TUNEL staining channels shown. Scale bar = 100 µM.