Knockdown of Sec16 causes early lethality and defective deposition of the protein Rp30 in the eggshell of the vector Rhodnius prolixus

Abstract

In nearly every species of insect, embryonic development takes place outside of the mother's body and is entirely dependent on the elements that the mother had previously stored within the eggs. It is well known that the follicle cells (FCs) synthesize the eggshell (chorion) components during the process of choriogenesis, the final step of oogenesis before fertilization. These cells have developed a specialization in the massive production of chorion proteins, which are essential for the protection and survival of the embryo. Here, we investigate the function of Sec16, a protein crucial for the endoplasmic reticulum (ER) to Golgi traffic, in the oocyte development in the insect Rhodnius prolixus. We discovered that Sec16 is strongly expressed in vitellogenic females' ovaries, particularly in the choriogenic oocyte and it is mainly associated with the FCs. Silencing of Sec16 by RNAi caused a sharp decline in oviposition rates, F1 viability, and longevity in adult females. In the FCs, genes involved in the unfolded protein response (UPR), the ubiquitin-proteasome system (UPS), and autophagy were massively upregulated, whereas the mRNAs of Rp30 and Rp45-which code for the two major chorion proteins - were downregulated as a result of Sec16 silencing, indicating general proteostasis disturbance. As a result, the outer surface ultrastructure of Sec16-silenced chorions was altered, with decreased thickness, dityrosine crosslinking, sulfur signals, and lower amounts of the chorion protein Rp30. These findings collectively demonstrate the critical role Sec16 plays in the proper functioning of the FCs, which impacts the synthesis and deposition of particular components of the chorion as well as the overall reproduction of this vector.

Keywords: Sec16; chorion; follicle cells; oogenesis; secretory pathway.

FIGURE 1

Maximum likelihood phylogenetic analysis of proteins with PF12931 domain across species. Sequences were aligned using the MUSCLE tool and the phylogenetic tree reconstruction was made using the maximum likelihood method. The tree was designed with 1000 replicates of rapid bootstrap statistics. Dark blue represents Sec16 proteins from different species. Light blue represents Sec31 proteins from different species. R. prolixus Sec16 is dot-marked.

FIGURE 2

Sec16 is highly expressed in the ovary and is present in all stages of oogenesis in vitellogenic females. (A) Representative scheme of the Sec16 protein from R. prolixus, D. melanogaster, and H. sapiens and their conserved domains Sec16_C (pfam12931), the binding domain of the Sec23 protein. (B) RT-qPCR showing the relative expression levels of Sec16 in different organs of the adult female. MG: Midgut; FB: Fat body; OV: Ovary. (n = 5–6). One-way ANOVA. (C) RT-qPCR showing the relative expression levels of Sec16 throughout all stages of oogenesis. (n = 4–8). One-way ANOVA. Graphs show mean ± SEM. **p < 0.01, ***p < 0.001.

FIGURE 3

Silencing of Sec16 results in reduced lifespan, impaired weight loss and higher levels of Vg in the hemolymph. (A) RT-qPCR shows the Sec16 knockdown efficiencies in the different organs 7 days after the blood meal. MG: Midgut; FB: Fat body; OV: Ovary. (n = 4–6). t-test. (B) Survival rates of control and dsSec16-injected females (n = 20). Log-rank (Mantel-COX) test. (C) Effects of Sec16 silencing on female digestion, showing their weight during the digestion cycle (n = 20). Two-Way ANOVA. (D) Control and silenced hemolymph protein quantifications 7 days after the blood meal. (n = 12). t-test. (E) 10% SDS-PAGE of the hemolymphs. Arrowheads point to the vitellogenin apoproteins. (n = 3). (F) Densitometry of the Vg apoproteins apoVg-I, apoVg-II and apoVg-III shown in (E) (n = 3). t-test. †: Indicates the death of all individuals. AU: Arbitrary units. Graphs show mean ± SEM. *p < 0.05, ****p < 0.0001.

FIGURE 4

Silencing of Sec16 results in abnormal oogenesis. (A) Representative images of ovarioles. Scale bar: 1 mm. (B) Representative images of cross-sections from silenced and control choriogenic oocytes under the light microscope. Scale bar: 50 μm. (C) Choriogenic oocytes protein quantifications, dissected 7 days after the blood meal. (n = 6). t-test. (D) 10% SDS-PAGE of choriogenic oocytes. Arrowheads point to the vitellin apoproteins. (n = 3). (E) Densitometry of the Vt apoproteins apoVt-I, apoVt-II and apoVt-III shown in (D) (n = 3). t-test. (F) Oviposition of control and silenced females during the gonotrophic cycle. (n = 20). Two-Way ANOVA. (G) F1 total hatching rates after Sec16 silencing. (n = 20, with 884 eggs dsMal and 122 eggs dsSec16). t-test. (H) Phenotypic distribution of control and silenced eggs and their respective viabilities. (n = 20). (I) Representative images of eggs laid 24–96 h after oviposition. Scale bar: 0.5 mm. AU: Arbitrary units. Graphs show mean ± SEM. *p < 0.05, ****p < 0.0001.

FIGURE 5

Sec16-silenced FCs upregulate UPR effectors and genes of the autophagy and UPS machinery while reducing the expression of the main chorion proteins Rp30 and Rp45. (A,B) Silencing of Sec16 results in the upregulation of genes related to UPR, autophagy, and UPS machinery in the FCs. (A) RT-qPCR showing the upregulation of the UPR sensors IRE1α and PERK and some isoforms of the chaperones BIP and PDI. (n = 3). t-test. (B) RT-qPCR showing the upregulation of the autophagy genes ATG3 and adaptor protein p62/SQSTM1 and the E1 and E2 enzymes of the ubiquitin-proteasome system. (n = 3). t-test. (C) RT-qPCR showing the downregulation of the main chorion proteins after Sec 16 silencing in the choriogenic oocyte, a follicle that contains the tissue responsible for chorion synthesis. The Vitellarium represents all the developmental stages of oogenesis except the choriogenic oocyte. (n = 3–4). t-test. Graphs show mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

Sec16 deficiency results in abnormal chorion biogenesis. (A) SEM images evidencing the abnormal ultrastructure of the operculum and the exochorion surface and the reduced chorion thickness in Sec16 eggs up to 72 h after oviposition. Representative images of 4 different images. Scale bar: 200, 20, and 25 μm, respectively. (B) Mean thickness of the chorion of control and silenced eggs. (n = 12, with 3 measurements of different regions of each image). t-test. (C,D) Silencing of Sec16 decreased the tyrosine crosslinking rates in the eggs. (C) Representative images of the dityrosine fluorescence in eggs chorion under UV light. Representative images of 5 different control and silenced images. Scale bar: 0.5 mm. (D) Mean of dityrosine fluorescence quantification using ImageJ software. (n = 5). t-test. (E,F) Sec16 deficiency results in chorion defective elemental composition. (E) Effect of silencing on the distribution of elements on the exochorion surface. (F) Signals obtained from the spectra of the different elements. The right figure shows the spectra in an expanded form. Representative images of 5-6 different control and silenced images. Blue arrows indicate the element sulfur. (G,H) Sec16 silencing reduces the level of Rp30, 1 of the main chorion proteins. (G) 10% SDS-PAGE of chorion proteins from 0 to 72 h eggs, extracted in urea. Representative image of 3 different gels. Arrowheads indicate Rp30, Rp45, Rp100, and Rp200. (H) Densitometry of the protein bands indicated in G performed using ImageJ software. (n = 6). t-test. AU: Arbitrary units. Graphs show mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 7

Schematic diagram summarizing the effects of silencing Sec16 in the chorion formation in R. prolixus. In the control condition (dsMal), during the choriogenesis process, the FCs act in the synthesis and secretion of the chorion components. Within the FCs, chorion proteins are directed from the ER to the Golgi apparatus via COPII vesicles, proceeding through the entire secretory pathway. This pathway allows the correct export of the chorion components to the extracellular space and their deposition over the oocyte cell membrane, enabling the production of eggs with a rigid eggshell capable of protecting the developing embryo. On the other hand, in the silenced condition (dsSec16), our data suggest that Sec16 silencing results disturbed proteostasis (ER stress) in the FCs under due to the hindered exportation of the cargo through COPII vesicles. This disruption is evidenced by the upregulation of genes involved in cellular proteostasis pathways (UPR, UPS and autophagy) and the expansion of ER lamellae compared to the control condition, consistent with previous findings by Bomfim and Ramos, 2020. As a result, the faulty functioning of the FCs leads to the production of malformed chorions, with reduced thickness and lower amounts of the Rp30 protein, culminating in reduced embryonic viability rates. In addition, the silencing of several other genes has been previously reported to culminate in chorion malformations, indicating that the coordination of choriogenesis by the FCs is a complex and sensitive process in R. prolixus. ER: Endoplasmic reticulum; LD: Lipid droplets; M: Mitochondria; N: Nucleus.