Atretic follicles were identified using several visual cues including bright red NR-stained vesicles within the follicular epithelium, asynchronous and smaller and/or rounder follicles, and a loss of clear follicular cell types ( Figure 3A ). Atresia was observed from 12-48 hpbm and peaks with ~17% of follicles undergoing resorption between 24-36 hpbm ( Figure 3B ). All atretic follicles observed were equivalent or smaller in length to the average 12 hpbm follicle ( Figure 3C ).

To assess whether PCD was occurring in atretic follicles, TUNEL staining of was used to reveal evidence of DNA fragmentation. TUNEL staining evident in follicular epithelial cells of atretic follicles ( Figure 4 ). Neither the oocyte or nurse cell nuclei were TUNEL positive in early atretic follicles ( Figure 4A ) or more advanced atretic follicles ( Figure 4B ). This result emulates that of NR stained atretic follicles (compare Figures 3A , 4B ).

The progression of NCD was measured by NR and acridine orange staining ( Figures 5 ; S5 ). NCD first appears with permeabilization of individual nurse cell nuclear envelopes at 46 hpbm ( Figure 5A ) with 50% of follicles undergoing NCD by 60 hpbm in 3-day old adults ( Figures 5B ; S5A ). In older mosquitoes (5-days old at the time of bloodfeeding) follicles undergo NCD earlier with 50% undergoing NCD by 52 hpbm ( Figure S5B ). By 72 hpbm, nurse cells are no longer distinguishable. During the process of NCD, nurse cells condense and stain bright crimson with NR as a marker of acidification. This staining is first localized to the nurse cells (see Figure 1E ), but nurse cell remnants are later visible on the follicular epithelium ( Figures 1F, G ).

NCD is asynchronous both between and within follicles, with follicles displaying anywhere from 1-7 apoptotic nurse cells from 46-72 hpbm ( Figures 5C–E ). Over time, progressively more nurse cells undergo NCD and become increasingly compartmentalized as contents are lost to the oocyte or phagocytosed by the follicular epithelium ( Figures 1E–G , 5D, E ). The compaction of nurse cells is coupled with binucleation and a shift of green to red staining and nuclear fragmentation indicating acidification of cellular microvesicles (stained by acridine orange) or release of RNA from the nucleus ( Figures 5C–E ). Compaction, fragmentation of nuclei, and staining with acridine orange indicates apoptotic type death (16, 38). To confirm apoptosis type DNA fragmentation, a sign of late apoptosis, TUNEL staining was performed at 60 hpbm on follicles with normal morphology and revealed a portion of TUNEL positive nurse cells in 2/3 follicles displayed ( Figure 6A ). TUNEL, NR, and acridine orange staining were in agreement in that NCD occurs asynchronously.

To determine whether cells of the follicular epithelium undergo apoptosis in order to be removed TUNEL staining was performed at 60 hpbm and 96 hpbm during mid- and late-oogenesis respectively ( Figures 6A, B ). Apoptotic DNA fragmentation was noted in a small number of scattered cells in both mid- and late-stage follicles, but the majority of nuclei appear normal and do not stain with TUNEL compared to the positive control ( Figure 6C ). In these cells actin is also noticeably uncoordinated. Whether actin dysregulation is a function of apoptosis or caused by mechanical damage to individual cells was not determined. To detect whether the epithelium undergoes apoptosis prior to or post oviposition, TUNEL staining was performed on segmented ovaries at 120 hpbm but no cells appeared TUNEL positive at this time either pre-, during-, or post-oviposition ( Figures S6A–C ). Removal of the follicular epithelium is thought to be driven by apoptosis prior to removal from the egg at the anterior pole after deposition of chorion in higher flies (13, 39). By contrast, while some cells undergo apoptosis asynchronously in Ae. triseriatus ( Figures 6A, B ), the entire epithelium containing many intact cells is dislodged from the follicle prior to oviposition in what appears to be a coordinated mechanism in Ae. triseriatus (see Figure 1H ). Follicular epithelial cells are apparent in cases after oviposition between 120-144 hpbm ( Figure S4B ) but are not visible in others where secondary follicles are developing ( Figure S4C ) and so may undergo cell death or become phagocytosed following oviposition.

Oogenesis events in an anautogenous mosquito precede the bloodmeal; in the previtellogenic phase (stage G), follicular progenitor cells replicate in the germarium then are separated in ovarioles (43). Primary follicles are the distal-most follicles of each germarium. These develop a follicular epithelium layer, and the oocyte becomes distinguishable from the nurse cells (Stage Ia-Ib). Next, the oocyte becomes more recognizable with the formation of lipid droplets, while the oocyte nucleus and nucleolus remain visible by reflected light (stage IIa-IIb). Throughout the previtellogenic phase, follicles are resistant to NR staining. We find that immediately following a bloodmeal, Ae. triseriatus follicles are in previtellogenic stages Ib-IIb. Herein, follicles appear clear and spheroid with oocyte nuclei visible clearly at 150x magnification and no positive staining with NR in the oocyte ( Figure S1A ). By 4 hpbm, most follicles remain at previtellogenic stages Ib-IIb and appear largely unchanged in size and shape ( Figures S1B ; 2A ).

In anautogenous oogenesis, ingestion of a bloodmeal stimulates release of ovary ecdysteroidogenic hormone (OEH) and insulin-like peptides (ILPs) from neurosecretory cells in the brain that stimulate ecdysteroid hormone (ECD) production in the ovaries (22, 44–47). At the same time, blood cholesterol is absorbed into follicle epithelial cells is converted to ecdysone that stimulates vitellogenin production in the fat body (15). The release of OEH and ILPs, and interaction with other pathways such as the Target of Rapamycin (TOR) also regulate midgut gene expression to digest the bloodmeal and initiate yolk protein production in the fat body and uptake of yolk proteins in the oocyte in lipid vesicles (15, 22, 48, 49). OEH, ILPs and ECD therefore act as the gate between the previtellogenic phase and the initiation phase ( Table S1 ), which is marked by NR staining of lipid vesicles in oocytes ( Figure 1A ). In 3-day old Ae. triseriatus, no follicles stain positive with NR at 0 hpbm, indicating that no follicles pass the initiation gate before or immediately following a bloodmeal, and that follicles require time for yolk protein production and uptake from the fat body ( Figure S1E ). This is supported by the presence of clearly visible oocyte nuclei, even at 4 hpbm, characteristic of Stage IIb and Christophers’ Stage 2 ( Table S1 ; Figures S1A, B ). Occlusion of the oocyte nucleus, another mark of stage IIIa, occurs between 4 - 12 hpbm as yolk protein uptake continues and 100% of follicles stain positive with NR ( Figures 1A, B ; S1E ). In older individuals (5 day post-eclosion AKA 5-day old adults) follicles were larger at 0 hpbm and stained positive with NR at 0 and 4 hpbm ( Figures S1C, D ), indicating that some vitellogenesis and receptivity to yolk protein uptake occurs prior to or immediately following a bloodmeal. The oocyte nuclei of 5-day-old adults were still visible in many follicles, despite positive NR staining ( Figures S1C, D ). Therefore, it may be that NR staining is not strictly indicative of stage IIIa in Ae. triseriatus if oocyte nuclei are still visible as this is a hallmark of phase IIb ( Table S1 ). Ovaries from older adults (up to 11-days) were assessed but were indistinguishable from 5-day old adults.

Oocyte content was tracked as a percentage of overall follicular area as a key indicator of trophic phases ( Figure 1C ). At 12 hpbm, visible oocyte content reaches 43% of the follicle, increasing to 60% by 18 hpbm ( Figure 2C ). Because the cutoff for stage IIIa is defined as 50% oocyte content, stage IIIb must begin between 12 and 18 hpbm. Stage IIIb marks the start of the trophic phase, which is not defined by a change in shape, but an increase in oocyte content from 50-75% alongside an increase in follicle size. Another marker of stage IIIb in Ae. aegypti is peak follicular atresia, which may in this case act as a secondary stage marker (24). Here we quantified atretic follicles and discovered a distinct peak from 24-36 hpbm ( Figure 3B ). Based on size increase, lack of shape change, and peak atresia, stage IIIb occurs in Ae. triseriatus at 18-36 hpbm.

At the point of transition to trophic stage IVa, Ae. triseriatus deviates from the criteria set forth previously for other mosquitoes. Stage IVa is characterized by continued growth, change in shape of the follicle and thinning of the follicular epithelium, up to 90% oocyte content, and intact nurse cells (see Supplemental Table 1 ). In Ae. triseratus, the criteria for trophic stage IVa for oocyte content are met at 18-24 hpbm, but follicle size continues to increase without a shape change until 48-60 hpbm ( Figures 1A, B ). The morphological criteria also fit using this timeframe because follicle growth continues up beyond 60 hpbm ( Figure 2A ) and follicular epithelium thinning starts between 48-60 hpbm ( Figure 2B ). We observed that stage IVa occurs in the window of follicular growth between two major cell death events: atresia (during stage IIIb) and NCD (during stage IVb). Since peak atresia ends at 36 hpbm ( Figure 3B ) and NCD does not occur in the majority of follicles until 60 hpbm ( Figure 5B ), stage IVa occurs between 36 and 60 hpbm for the first gonotrophic cycle.

The end of the trophic phase marks the transition to stage IVb. At this point, the oocyte encompasses almost 100% of the follicular interior and the follicle assumes the shape of the mature egg. These criteria set stage IVb between 48-72 hpbm (compare Figures 1E–G , 2A, B ). Similar to late oogenesis in Drosophila, nurse cells undergo NCD when the oocyte nears maturity (36, 50). NCD is a hallmark of stage IVb and is evidenced by bright crimson staining of nurse cells using NR ( Figures 1E, F , 5A ). This event is first observed at 48 hpbm in Ae. triseriatus, but does not peak until 60 hpbm ( Figure 5B ). Finally, Clements & Boocock note that in stage IVb, the “follicle assumes the shape of the mature oocyte, or almost so, but has not reached full length” (24). In Ae. triseriatus, follicles reach maximum length at 72 hpbm.

The post-trophic stage marks the final events of oogenesis that occur in rapid succession. Here follicles reach maximum length, shed the follicular epithelia, and chorionic structures become visible. In this study, at 72 hpbm, 100% of nurse cells were degraded indicating the end of the trophic phase ( Figure 5B ). Follicles at this time also reach a size maximum and thinning halts ( Figures 2A, B ). By 96 hpbm, chorionic structures are also visible and the follicular epithelium detaches, revealing the fully developed oocyte ( Figures 1F, G ). We consistently observed the follicular epithelium rolling onto itself and down the length of the follicle while still in the ovariole.

During the first gonotrophic cycle, secondary follicles grow and mature from stage G to initiation phase II by 96 hpbm (compare Figures 1H ; S1A ). Prior to oviposition, secondary follicles do not pass into the initiation phase but stain with NR throughout ( Figure S1A ). Following oviposition, the primary follicle follicular epithelium remains in the ovary and is resorbed over ~24 hours ( Figures S1B, C ). Resorption of the follicular epithelium appears to be essential to clear the distalmost region the ovariole to allow secondary follicles to assume this position ( Figure S1B ). Immediately following oviposition, the follicular epithelium remains intact and secondary follicles do not pass into stage IIIa, but show characteristics of stage IIb ( Figure S1B ; Table S1 ). Resorption of the follicular epithelium is associated with the secondary follicles entering stage IIIa; as such, the follicular epithelium may provide nutrition sufficient for development of some secondary follicles without need for a second bloodmeal ( Figure S1C ).

In insects, atresia occurs during early-mid stages of oogenesis (28). The process necessitates termination of yolk deposition in the oocyte and complete degradation of the follicle. In other fly species (e.g., D. oleae and D. melanogaster) atresia occurs as a function of PCD in the form of apoptosis and/or autophagy (26–28, 53). In Ae. aegypti, atresia is noted during stage IIIa and peaks during stage IIIb between 25-30 hpbm (24). Our results show atretic follicles as early as 12 hpbm, which aligns with stage IIIa morphological data ( Figure 3B ). All of the atretic follicles observed in Ae. triseriatus were equivalent or smaller in length to the average 12 hpbm follicle ( Figure 3C ), which may indicate that a nutritional deficiency/sufficiency gate occurs at this time point or follicle size. Although atresia has not been used to define this stage in Ae. aegypti, it is informative in Ae. triseriatus. This may be especially useful for defining stage IIIb in Ae. triseriatus because the transition from IIIa to IIIb in this mosquito does not fit the criteria for other species ( Table S1 , section 3.3.3).

Interestingly atretic follicle oocytes and nurse cell nuclei did not stain with TUNEL, but the follicular epithelium did. This result emulates that of NR stained atretic follicles (compare Figures 3A , 4B ). Atresia may be localized or controlled by the follicular epithelium in Ae. triseriatus; this is congruent with atresia in Culex pipiens pallens wherein active caspases are restricted to the epithelial cells of atretic follicles (32). Similar results were seen in Plasmodium-infected Anopheles stephensi, wherein atretic follicles exhibited apoptosis mainly in the follicular epithelium (54). By contrast, in the higher flies, D. oleae, D. melanogaster, and Ceratitis capitate, apoptosis was evident in the nurse cell compartment of atretic follicles (20, 27, 36, 39).

Nurse cells are fundamental to oogenesis in many multicellular organisms and function in much the same way in flies as in Caenorhabditis elegans and Hydra (50). In Aedes species, each follicle contains a single oocyte and 7 nurse cells that provide cytoplasmic material including mRNA and protein directly to the oocyte via ring canals (43). After the oocyte has sufficiently grown and developed, the nurse cells degrade and die. In Drosophila, NCD starts with permeabilization of the nuclear membrane, followed by transportation or ‘dumping’ of cytoplasmic contents (including large amounts of RNA and protein) to the oocyte through the ring canals, and finally degeneration and apoptosis of the remaining cell (50, 55). To decipher the similarities of NCD between Drosophila and Ae. triseriatus, numerous staining techniques were employed. In D. melanogaster, acridine orange staining produces green/red nuclei during NCD, particularly in nuclei that are permeabilized as a function of an apoptotic event (38, 54). We witness the same permeabilization in nuclei which occurs sporadically between nuclei within and between follicles with anywhere from 1-7 nurse cells undergoing NCD at any given time from 46-72 hpbm ( Figure 5B ). Nurse cells decrease in size, and shift from green to red staining with acridine orange ( Figures 5D, E ). The decrease in nurse cell size is possibly due to cytoplasmic dumping, while the shift in color is either due to acidification of the cell during apoptosis, or increase in RNA production based on the staining characteristics of acridine orange (56). Additionally, red-stained vesicles were often seen dispersing from the nurse cell compartment, suggesting that acridine orange may mark RNA or acidic vesicle movement from the nurse cell compartment to the oocyte. Nurse cells undergoing NCD also displayed increased DNA fragmentation in TUNEL assays and as such are likely undergoing apoptosis ( Figure 6A ). Acidification of microvesicles within the nurse cells is likely driven by autophagy, which would explain the shift from green to red staining in nurse cells undergoing NCD. However, further studies are required to confirm whether autophagy drives NCD along with apoptosis in Ae. triseriatus in the same way as observed in higher flies (35).

The epithelium is a dynamic and multifunctional cell layer and constitutes a significant area (between 15 and 30%) of the overall follicle size ( Figure S3 ). Ultimately, the epithelium deposits a protective vitelline chorion layer (43), and at the post trophic phase (stage V), the epithelium is sloughed to release the mature oocyte ( Figure 1H ). This phenomenon is different from Drosophila and other higher flies where follicular epithelial cells undergo apoptosis while attached to the developing follicle, starting from the anterior pole, are sloughed away from the follicle, and then engulfed by epithelia of the oviduct (36, 57). To investigate whether these cells undergo apoptosis while still associated with the follicle, TUNEL staining was performed ( Figure 4 ; Figure S6 ). The results show that only a small number of epithelial cells stain with TUNEL, with the remaining displaying large, fully formed nuclei typical of healthy cells, but are sloughed away from the developed stage V oocyte prior to oviposition. The removal of viable follicular epithelial cells in Ae. triseriatus indicates that there is more research to be done to reveal the mechanism behind epithelium death and cell sloughing. Additionally, TUNEL positive follicular epithelial cells could reflect phagocytosed remnants of degraded nurse cells, as is seen in D. melanogaster (39). After removal from developed primary follicles, follicular epithelial cells stained crimson with NR, but this staining may be remnant material from NCD and not a result of acidification or chromatin condensation (see Figure S4 ). Positive control TUNEL stain was observed in primary follicle epithelium pre- and post- sloughing as well as secondary follicle and the ovarian sheath epithelia ( Figure S6 ). Therefore, epithelial cells do not appear to be resorbed during the first gonotrophic cycle and may be retained for longer periods in Ae. triseriatus. Interestingly, at 120-144 hpbm secondary follicles were observed developing past the previtellogenic phase into stage IIIa with no observable remnants of the primary follicle epithelium (see Figures S4B, C ) and so it is likely that nutrients from the follicular epithelia are utilized to initiate vitellogenesis production and growth of secondary follicles without a secondary bloodmeal. However, whether the oviduct absorbs the follicular epithelium, and the timing and appearance of follicular epithelial cell death remains to be discovered in Ae. triseriarus.