Spatio-temporal patterns of ovarian development and VgR gene silencing reduced fecundity in parthenogenetic Artemia

The halophilic zooplankton brine shrimp Artemia has been used as an experimental animal in multidisciplinary studies. However, the reproductive patterns and its regulatory mechanisms in Artemia remain unclear. In this study, the ovarian development process of parthenogenetic Artemia (A. parthenogenetica) was divided into five stages, and oogenesis or egg formation was identified in six phases. The oogenesis mode was assumed to be polytrophic. We also traced the dynamic translocation of candidate germline stem cells (cGSCs) using EdU labelling and elucidated several key cytological events in oogenesis through haematoxylin and eosin staining and fluorescence imaging. Distinguished from the ovary structure of insects and crustaceans, Artemia germarium originated from ovariole buds and are located at the base of the ovarioles. RNA-seq based on five stages of ovarian development identified 2657 upregulated genes related to reproduction by pair-to-pair comparison. Gbb, Dpp, piwi, vasa, nanos, VgA and VgR genes associated with cGSCs recognition and reproductive development were screened and verified using qPCR. Silencing of the VgR gene in A. parthenogenetica (Ap-VgR) at ovarian development Stage II led to a low level of gene expression (less than 10%) within 5 days, which resulted in variations in oogenesis-related gene expression and significantly inhibited vitellogenesis, impeded oocyte maturation, and eventually decreased the number of offspring. In conclusion, we have illustrated the patterns of ovarian development, outlined the key spatio-temporal features of oogenesis and identified the negative impacts of VgR gene knockdown on oogenesis using A. parthenogenetica as an experimental animal. The findings of this study also lay a foundation for the further study of reproductive biology of invertebrates.


Introduction
Reproductive development is vital for the development and propagation of multicellular organisms [1].Sequential events during ovarian development and oogenesis, also known as egg formation, are critical for female reproduction [2].Egg formation in oviparous females have been well studied, especially in classical 'model' organisms and some insects [3,4].However, the subject of evolutionary developmental biology (evo-devo) has become an emerging field that requires a broader picture in the context of high biodiversity [5,6].Ovarian development in bisexual species generally includes mature follicle generation, oestrogen secretion, egg production, fertilization and other important processes [7] that are accompanied by successive cytological events.In Drosophila, oogenesis requires extensive interactions between somatic and germline stem cells, and these developmental events occur in the ovaries [8].Oogenesis predominantly includes primordial germ cell (PGC) formation and oocyte development, differentiation and maturation.These are controlled and regulated by relevant genes and signalling pathways [9].As a key subject in developmental and reproductive biology, oogenesis has been extensively studied in mammals, insects and crustaceans [10][11][12].However, to date, there has been relatively little research on the patterns of reproductive processes and molecular mechanisms in invertebrates, especially for species living in extreme habitats such as those that are hypersaline or hypoxic.To date, this has resulted in a relatively limited understanding of reproductive evolution.
Hypersaline environments have provided an extraordinary habitat for all domains of life on Earth since the Precambrian [13].The brine shrimp Artemia is affiliated with Crustacea, Branchiopoda and Anostraca, thriving in hypersaline water bodies with salinity up to 20%.Artemia has several biological advantages and unique life phenomena, such as a transparent body, asexual and bisexual reproduction modes, oviparity and ovoviviparity breeding strategies, and diapause and de-diapause life cycles [14].Therefore, Artemia has been used as an experimental animal in environmental toxicology, oncology, epigenetics and other research fields [15][16][17].Compared to bisexual Artemia, parthenogenetic Artemia (A. parthenogenetica) imposes a special reproduction mode.The female accomplishes a process of sexual reproduction in which the unfertilized egg develops without the contribution of the male gamete [16].This ensures the production of offspring that develops from unfertilized embryos as well repeatability of the experiments.These offspring are considered 'full clones' with the same genotype and are genetically identical to their mother [18,19].For Artemia, the subjects of biological research mainly focuses on diapause and de-diapause mechanism [20], viviparous and ovoviviparous reproduction regulation [21] and physiological response to the toxicological exposure [22,23].
In oviparous animals, vitellogenesis is a prerequisite for oviposition and embryo development [24].Juvenile hormone (JH) signalling plays a crucial role in insect reproduction [25,26].But whether the arthropod-specific sesquiterpenoid (JH) is present in crustaceans, and the member of downstream gene and the molecular mechanisms remain elusive.Vitellogenin (Vg) is synthesized in the hepatopancreas of crustaceans [27] and the fat body (FB) of insects [28].Vg is transported through the blood, binds to vitellogenin receptors (VgRs) located on the plasma membrane of oocytes to form the Vg/VgR complex, and enters the oocyte via endocytosis [29].VgR knockout or mutation can inhibit oocyte maturation in insects or cause sterility in birds, indicating that VgR plays a crucial role in ovarian development in oviparous animals [30,31].To date, researchers have focused on other genes that play key roles in oogenesis, such as Dpp (Decapentaplegic)and Gbb (Glass bottom boat) in the BMP signalling pathways, as well as vasa (Vasa intronic), nanos (Nanos homologue) and piwi (P-element-induced wimpy) in reproductive stem cells.Among these, Dpp and Gbb mainly promote the growth of follicular cells or regulate the synthesis of JH by inhibiting the gene encoding JH acid O-methyltransferase [32,33].Silencing of vasa, a marker of reproductive stem cells, changed the morphology of ovaries and oocytes and reduced the number of eggs in the Japanese blood-sucking worm Schistosoma japonicum [34].nanos and piwi are important in regulating the expression of germline stem cells [35].To date, no studies have been conducted on the regulatory genes, pathways, functions and molecular mechanisms of the genes involved in A. parthenogenetica oogenesis.
Our study has reported the morphological and tissue characteristics of the ovarian development process, clarified the cytological details of oogenesis.We also screened candidate genes that may be involved in the ovarian development and oogenesis in Artemia with a focus on the function of the Ap-VgR gene in the oogenesis process.The aim of this study is to provide insights for understanding the mechanism of ovarian development in reproductive biology and to establish a platform for studying the life processes of invertebrates.

Artemia rearing
Artemia parthenogenetica cysts from Aibi Lake, China, were obtained from the Asian Regional Artemia Reference Center (ARARC), Tianjin University of Science and Technology, China.Approximately 0.2 g cysts were incubated in a conical tube containing 200 ml dilute brine water with a salinity of 3% under constant hatching conditions at 28°C with 2000 Lux illumination and continuous aeration.After 24 h, Artemia nauplii were collected and transferred to a rectangular plastic tank containing 16 l of diluted brine with a salinity of 7%.The initial density was 130 individuals l −1 .The animals were fed twice daily with Chlorella sp.The water was renewed once weekly.The ovary morphology of Artemia was observed under a stereoscope (SZ680, China) when the oocyst primordia appeared.

HE staining
To examine the histological and cytological features in the process of ovarian development, A. parthenogenetica individuals representing five developmental stages were collected.

Immunofluorescence antibody labelling
To describe the dynamic migration pathway of germline stem cells during oogenesis, A. parthenogenetica ovaries were dissected using forceps and incubated with a mixture of primary antibodies in a cell incubator (Corning) for 2 h.The mixture comprised 2 × EdU working solution (Thermo Fisher, Lot: 2261449), DMEM (Gibco, Lot: 1897090), penicillin, streptomycin and neomycin (Gibco, Lot: 1894168).Tissues were fixed in 4% paraformaldehyde for 2 h.After rinsing with PBS, the tissues were sealed with 3% BSA (Thermo Fisher Scientific, Lot: SF248474) for 20 min.0.5% Triton 100 was used to increase the permeability of the antibodies to the cell membrane.Secondary antibodies conjugated with Alexa Fluor 488 (Thermo Fisher, Lot: 2261449) were incubated (1 : 500, v/v) with the tissues for 2 h under dark and moist conditions.Finally, the samples were incubated with Hoechst 33342 at 28°C for 30 min at a ratio of 1 : 2000 (v/v).Anti-fluorescent quenching agent was added after cleaning.Images were acquired using a laser confocal microscope (Zeiss 980, Germany).

RNA extraction and illumina sequencing and data analysis
Artemia ovaries at five developmental stages were dissected in cold liquid nitrogen.Thirty individuals at the same developmental stage were pooled.RNA extraction was performed using RNAiso Plus reagent (TaKaRa, AKF0727A).Transcriptome library construction and subsequent sequencing analyses were performed by Novogene Technology Co. Ltd., China.Genbank accession number (SAMN36887526) of all used sequences can be found in NCBI.
The original data obtained from high-throughput sequencing were filtered.Functional unigenes were annotated using seven databases, including Gene Ontology (GO; http://www.geneontology.org/)and the Kyoto Encyclopaedia of Genes and Genomes (KEGG; https://www.kegg.jp/).The structural domains of the genes were analysed using SMART.A phylogenetic tree was constructed by the maximum-likelihood method, using MEGA7 with 1000 bootstrap replicates.

RT-qPCR analysis
To explore the spatio-temporal expression patterns of reproductive genes in Artemia, total RNA was extracted from the target tissues, namely those of the intestine, cerebral ganglion and ovary, at five developmental stages using RNAiso Plus reagent (TaKaRa, Lot: AKF0727A).cDNAs was synthesized from 1 µg of total RNA (Thermo Fisher Scientific, Lot: A48571).RT-qPCR was performed using TB GreenPremix Ex Taq II (TaKaRa Lot: AL51019A), and β-actin was used as a reference gene.Primers were designed using Primer 5 software (electronic supplementary material, table S1).
Each reaction contained three technical replicates and a template-free control.

RNAi and phenotypic analysis
Microinjection needles were prepared using a micropipette puller (Narishige PC-10, Japan) and 1.0 mm capillary tubes (VitalSense Scientific Instruments Co., Ltd.Lot: B100114N).Double-stranded RNA (dsRNAs) of VgR and EGFP were synthesized using the T7 RiboMAX Express RNAi System (Promega, USA).The primers used for dsRNA synthesis have been summarized in electronic supplementary material, table S2.After anesthetisation on ice, approximately 2000 ng of dsRNA was injected into the ventral side of the second abdominal segment at ovarian development Satge II using a microinjector (Nikon Eclipse Ti, Japan) fitted with an injection needle.The Artemia in the control group was treated with the same dose and volume of dsEGFP.Ovaries were sampled on the first, third and fifth days after dsRNA injection, and the total RNA was then extracted.The knockdown efficiency and gene variation were determined using qRT-PCR.
For phenotypic assessment, ovaries were collected when more than 80% of Artemia had reached the objective developmental stages.Ovary morphologies and cytological features were determined as described in §2.3.Morphological and cytological variations of the ovaries were observed under a fluorescence microscope (Nikon Ti-E, Japan).

Statistical analyses
The data are presented as mean ± standard deviation.Statistical significance was determined using one-way ANOVA followed by Duncan multiple comparisons at p < 0.05 and p < 0.01, respectively (SPSS26.0).Statistically significant differences between the two groups were inferred using a t-test.

Tissue characteristics in process of ovarian development
After approximately 24 h of hatching, most of the gastrula embryos of A. parthenogenetica developed into the Instar I nauplius stage which was considered a starting point for assessing the progress of ovarian development (figure 1, 0 dph, day post-hatching).Within 7-10 dph, approximately 63% of the individuals gradually entered the ovarian developmental period (Stage I), with a prominent pair of ovariole buds (dark arrows, in vitro) near the anterior ventral surface of the body.At 15-17 dph, the ovaries reached the mature period, of which approximate 51% individuals were further distinguished into pro-mature stage (II), metaphasismature stage (III) and anaphasis-mature stage (IV).The determination of the developmental stage was undertaken according to the ovarian morphology and characteristics such as the ovariole shape, location and features of oocytes (Stages II-IV).At 25-29 dph, the ovaries developed into the ovulatory period (Stage V), and the eggs resided in the oocyst with no longer transparent because of yolk granule accumulation.

Key cytological events of oogenesis
Based on the observation of the five stages of ovarian development of A. parthenogenetica, the morphological diagrams were drawn (figure 2a).On this basis, the whole process of egg formation could be further understood.At Stage III, the fifth phase oocytes were observed.As the ovaries developed, the number of opaque eggs increased.The egg chamber (EC) gradually formed in the ovarioles and contained the eggs that about to maturity.Follicular cells (figure 2b, III-2, FC, yellow arrow) were observed in Stage IV, with a row of single follicular cells (figure 2b, IV-2) surrounding the oocytes (figure 2b).At Stage V, the sixth phase oocytes were fully mature and entered the oocysts, which were enlarged black or grey cells.Before being released by the female, cytoplasmic cleavage occurs to form the multicellular egg (syncytium), which then develops into a gastrula embryo.

Spatial dynamic changes of candidate germline stem cells
As shown by the immunofluorescence images, the ovary of Artemia included four parts (figures 1 and 3): a pair of ovarioles (Or), an oviduct (Ovd), an oocyst (Oc) and an ovulatory hole (Oh).The FB was tightly embedded in the ovaries (figure 3b,c).The candidate germline stem cells (cGSCs) were identified using EdU labelling (figure 3a) during Stages I-III of ovarian development.Oocytes in other phases were confirmed based on these results (figure 2b) and their spatial position during ovarian development (figure 3).In Stages I and II of ovarian development, the cGSCs of Artemia were generated in the germarium (Gr), which contained dividing PGCs and oogonia produced from them.By contrast to the generation, maturation and migration routes of Drosophila GSCs, the Gr of Artemia was located at the proximal end of the oviduct (figure 3a, white dotted circles).The vitellarium (Vr) of Artemia was located royalsocietypublishing.org/journal/rsob Open Biol.13: 230172 at the distal end of the oviduct (figure 3c).As a pair of ovarioles fully developed, the oocytes increased in number and were surrounded by follicular cells that moved downward and migrated to the Vr, where they continued to develop and mature (figure 3a).Meanwhile, the Vr expanded to form a series of ECs in which oocytes were gradually deposited into the yolk proteins.To complete the reproduction, the eggs (gastrula embryos) either developed into nauplii via ovoviviparous reproduction, or were surrounded by a thick shell (ES, figure 2b, V-3) and entered a state of diapause royalsocietypublishing.org/journal/rsob Open Biol.13: 230172 (cysts, oviparous reproduction), which were released through the ovulatory hole (figure 1, triangular white arrow).

Transcriptome analysis of ovaries at five developmental stages
In total, 131 442 transcripts and 60 801 unigenes were obtained from the ovary tissues at the five developmental stages (electronic supplementary material, table S3).All the sequences were spliced using BLAST and compared with seven databases, including Pfam, Nr and Swissport, to obtain the corresponding annotation information.Annotations from databases are shown in electronic supplementary material, table S4 and figures S1-S5.
To clarify the gene expression profiles and molecular functions of key genes during ovary development process, 28 459 differential expressed genes (DEGs) were identified using transcriptome analysis at five ovarian developmental stages (figure 4a).On this basis, combined with the morphological changes in the five stages of ovarian development, We conduct a statistical analysis of DEGs in Stage IV versus Stage II of ovarian development.DEGs obtained in Stage IV versus Stage II ovarian development were statistically analysed.The volcano map shows the number of common and unique DEGs between the two groups (figure 4b).KEGG pathway analysis showed that 121 pathways were enriched in Stage IV compared to Stage II, and each pathway represented one or more biological processes.These pathways are relevant to progesterone-mediated oocyte maturation, the cell cycle and oocyte meiosis which can provide important information for subsequent studies on reproductive development (figure 4c).The results of DEGs analysis for other ovarian developmental stages are shown in electronic supplementary material, figures S4 and S5.

Expression profile of transcripts involved in reproduction-related genes
The FPKM value of each comparison group in the differential gene set of all comparison groups was calculated and these were used for hierarchical clustering analysis of the DEGs expression level (figure 5a).Some reproductive DEGs were screened, and the key genes in BMP signalling pathway (Dpp and Gbb) and JH signalling pathway (VgR and VgA) and other reproduction-related genes (vasa, piwi and nanos) were further verified by qRT-PCR (figure 5b).This showed royalsocietypublishing.org/journal/rsob Open Biol.13: 230172 that the expression profiles of these genes were consistent with transcriptome sequencing results (figure 5c).At all sampling times, transcript expression levels varied among the seven genes.However, a higher gene expression level of VgR, VgA, Dpp and Gbb were obtained, peaking at Stage III and/or Stage IV ( p < 0.05 or p < 0.01).The expression profile of these genes provided clues and a basis for implementing Artemia RNAi manipulation when appropriate.

Structure and phylogenetic analysis of VgR in A. parthenogenetica (Ap-VgR)
In line with the VgRs of other crustaceans [36], Ap-VgR contained three representative conserved modular elements of the low-density lipoprotein receptor (LDLR) superfamily (figure 6a).The ligand-binding domain (LBD) mediates receptor binding to ligands and contains Class A repeats (LDLa).The EGF-precursor homology domain (EGFD) mediates dissociation of receptor and liganded, and consists of YWXD motifs and epidermal growth factor (EGF) repeats.At the carboxyl terminus, transmembrane domain (TM) formed a transmembrane α-helix, which anchored the receptor to the plasma membrane.However, Ap-VgR does not contain an O-linked sugar domain (OLSD).These results suggest that Ap-VgR may regulate Vg transition into oocytes in a pattern similar to insect VgR.
The GenBank accession numbers of the proteins are shown in electronic supplementary material, table S5.Phylogenetic analysis showed that VgRs from various oviparous species were grouped into four clades affiliated with vertebrates, arthropods, mollusc and nematodes, and Ap-VgR was fitted into a clade of crustaceans and was more closely related to Daphniidae (figure 6b).

Expression profile of corresponding genes after
Ap-VgR slicing Ap-VgR transcript expression was detected in the intestine, cerebral ganglion and ovary of Artemia using qPCR (figure 7a).The results showed that Ap-VgR was specifically expressed in the ovary but was hardly detected in the other two tissues.The expression level of Ap-VgR increased continuously and reached the highest at ovarian developmental Stage IV.These results were consistent with the trends observed in the RNA-seq data.In this study, RNAi assays were conducted to determine the functions of Ap-VgR in the oogenesis of Artemia, and dsRNA was injected at the pro-mature stage according to the transcriptome results (figure 4).Compared to the dsEGFP group (control), the level of Ap-VgR in the dsVgR-treated group decreased by 89.8%, 90.2% and 85.2% on the first, third, fifth day post injection, respectively (figure 7b).The effect of Ap-VgR RNAi on the expression of oogenesis-related genes VgA, VgB, Dpp and Gbb was studied further.Compared to the dsEGFP group, the expression of VgA was significantly increased by silencing Ap-VgR.The expressions of VgB and Gbb were downregulated, whereas that of Dpp was not significantly altered (figure 7c).

Phenotypes modulation of oogenesis after Ap-VgR knockdown
The ovaries of Ap-VgR knockdown individuals exhibited clear phenotypic changes (figure 8a).In vivo observations showed that egg development in the ovary was delayed by the third day after dsVgR injection.On the fifth day after  royalsocietypublishing.org/journal/rsob Open Biol.13: 230172

Osteichthyes
and vitellarium ( p < 0.05).This further demonstrated that the knockdown of Ap-VgR delayed ovarian development and ultimately led to reduced fecundity (figure 8b,c and table 1).On the third day post dsRNA injection, HE staining of the ovary showed no pronounced morphological changes in the follicular cells after Ap-VgR gene silencing, and all of them had formed unicellular eggs (figure 8d).Meanwhile, the yolk protein further accumulated and aggregated in the eggs to form a large number of yolk granules in the dsEGFP group (figure 8d).However, the eggs in the dsVgR group were deformed with less accumulation of yolk granules but were rich in yolk protein.On the fifth day after dsRNA injection, the accumulation of yolk granules in the dsEGFP group was complete and typical granular eggs were formed in the oocyst.When the VgR gene was knocked down, cleavage was achieved in advance with pronounced unevenness and a substantial number of vacuoles, indicating inferior egg quality.The number of eggs in the dsVgR group was significantly lower than that in the dsEGFP group ( p < 0.01) on both third and fifth day after ovulation period (figure 9 and table 1).

Discussion
The continuation of life from generation to generation through reproduction and development is the biological basis for the proliferation of life [37].The biological advantages of A. parthenogenetica, such as colonial offspring production, relatively short lifespan and reproductive cycle, make it more suitable for the study of reproductive processes and molecular mechanisms [38,39].In the present study, the spatio-temporal features of ovarian development and oogenesis in A. parthenogenetica were clarified by means of in vivo and in vitro approaches.The FB tissue was first identified in Artemia, which is assumed to play a coincident role in promoting vitellogenesis in the fat bodies of insects and the hepatopancreas of crustaceans [40,41].The FB and the hepatopancreas provide Vg and promote its binding to VgRs.
We found significant differences in the GSC generative sites and migration pathways between A. parthenogenetica and Drosophila [4,8].Upon complete ovariole development, GSCs of Drosophila generated from the germarium near the terminal filament [42], whereas the oogonia of A. parthenogenetica originate from a pair of ovariole buds.Along with ovarian development, cGSCs population labelled by EdU migrate in the form of a cell mass to the corresponding sites where niches regulate egg formation (figures 1 and 3a).The formation of cell mass and the cGSCs niche in A. parthenogenetica may facilitate successive brood offspring production of Artemia, in favour of population sustainability in extreme environments.
Hormone-and gene-mediated regulation of reproductive development has been well studied in insects [43,44].However, to date, the mechanism of reproductive development in crustaceans in extreme environments has not been explored.The transcriptome platform is an important and convenient tool for providing data supporting the gene and gene network regulation [45,46].Our data have shown 28 459 DEGs related to ovarian development and oogenesis in Artemia, in which genes relevant to the cell cycle and oocyte meiosis, the signalling pathway of insulin and HIF were significantly expressed.This indicated that these genes may regulate and participate in key cyto-events in ovariole formation, cGSCs generation and migration, vitellogenesis and choriogenesis [47].In this study, Ap-VgR was screened and verified based on the DEGs in the transcriptome.VgR has been confirmed as a key receptor for vitellogenesis.This is fundamental for oocyte development in oviparous animals and serves as a key preparatory stage for establishing energy reserves and biomolecules for embryonic development [48,49].
We obtained the full-length cDNA of VgR in A. parthenogenetica and predicted its corresponding protein structural domains (figure 6a).In line with other members of the LDLR superfamily, Ap-VgR contains three highly conserved royalsocietypublishing.org/journal/rsob Open Biol.13: 230172 regions (LBD, EGFD and TM).However, similar to some insects, such as fire ants [50] and Drosophila melanogaster [51], there is a lack of an O-linked Carbohydrate Domain (OLSD) on the plasma membrane surface in A. parthenogenetica VgR.The tissue expression specificity of VgR is related to the presence of OLSD structures in VgR [52].In this study, the expression level of Ap-VgR without OLSD was significantly higher in the ovary than in other tissues, whereas the expression level of VgR in other species with OLSD was higher in non-ovarian tissues [53,54].The natural absence of VgR in OLSD can cause familial hypercholesterolaemia in humans [55].Therefore, the OLSD structure in VgR could be a potential marker of species reproductive specificity.RNAi of VgR suppresses Vg accumulation in the ovaries of the tiger shrimp P. monodon [56] and delays ovary maturation in the freshwater shrimp M. nipponense [57].However, the phenotypic details of VgR regulation during ovarian development are not well understood and remain controversial in different crustacean species [56,57].In the present study, the expression of Ap-VgR was specifically detected in the ovaries of Artemia (figure 7), which is consistent with most insects [58,59].Our results illustrated that the expression levels of Ap-VgR in the ovary increased continuously from ovarian developmental Stage I to Stage IV (figure 5b), indicating that the exogenous Vg absorption process is similar to that in other oviparous animals which exhibit exogenous vitellogenesis, such as fish [60] and insects [61].However, the expression levels of Ap-VgR decreased rapidly during the ovulatory period (Stage V), which was in accordance with the increased yolk consumption.These yolk reserves further sustain the survival of non-feeding Artemia nauplii for 3-4 d post-hatching.
In the present study, the potential molecular mechanisms underlying the onset of vitellogenesis, and offspring production were demonstrated through Ap-VgR regulation in Artemia.The abnormal eggs resulting from dsVgR injection and relatively low Ap-VgR expression over time indicate that Ap-VgR knockdown had a sustained inhibitory effect on yolk granule formation and led to a significant decrease in offspring production (figure 9).Multiple copies of the Vg genes were screened from the transcriptome and named using capital letters.Our results indicate Ap-VgR knockdown significantly increased VgA expression and decreased VgB expression.As has been reported in insects, VgR silencing may lead to the blocking of the expression of its ligand VgB [62].Meanwhile, the expression of VgA gene greatly increased, which seemed to compensate for Vg synthesis to rapidly restore its nutrient reserve [56].Another explanation is that different Vg gene family members are involved in different physiological functions, such as yolk protein formation and play non-nutritional roles [63].It is necessary to investigate the molecular characteristics of the Vg family as well as their functions in ovarian development, oogenesis, immune response or nutrient delivery.By contrast, Ap-VgR silencing reduced the expression of Gbb, a key gene in the BMP signalling pathway.BMP plays a regulatory role in the embryonic development of mice [64]; proliferation, differentiation and regeneration of germline stem cells in Drosophila [65]; and metamorphosis of insects [33].We have proposed that the decrease in Gbb expression affects development and offspring production by interfering with the normal proliferation and differentiation of germline stem cells.
In conclusion, our study has highlighted the processes and regulatory mechanisms underlying ovarian development and egg formation in A. parthenogenetica.The structure and function of Ap-VgR gene were more similar to those of insects but different from those of other crustaceans of the same class.It is likely that Artemia differentiated into the same class as crustaceans in ancient times, but some physiological characteristics and functions might be more similar to insects.Exploring the reproductive patterns and mechanisms of A. parthenogenetica may contribute to understanding the evolutionary route of animal reproduction from the sea to land, and how Artemia adapt to environmental changes and maintain population stability.With the publication of the genome of Artemia franciscana [66,67] and the completion of wholegenome sequencing of A. parthenogenetica (data not shown, ARARC sequenced), it is possible to find more accurate and detailed molecular evidence to interpret the role of Artemia in the evolution of species at the omics level.
Ethics.This work did not require ethical approval from a human subject or animal welfare committee.
All authors gave final approval for publication and agreed to be held accountable for the work performed therein.
Conflict of interest declaration.We declare we have no competing interests.Funding.This study was supported by the Open Fund of Key Laboratory of Experimental Marine Biology, Chinese Academy of Sciences (no. KF2019NO2) and the Open Project Program of Key Laboratory of Marine Resource Chemistry and Food Technology (TUST), Ministry of Education (no.EMTUST-22-04).
The cytological characteristics of the ovaries were shown by Hoechst and HE staining.The ovaries showed general bilateral symmetry throughout the ovarian developmental process (figure 2b, I-1,2, white dotted line).Based on the features of vitellogenesis and the accumulation and distribution of yolk granules observed under optical and fluorescence microscopy, six phases of oogenesis from oogonia to multicellular eggs were distinguished during egg formation (figure 2b, I-3, II-3,4, III-2,3, IV-2 and V-2,3).The oocyte was surrounded by follicular cells (figure 2b, IV-2, yellow arrows) and support cells (figure 2b, IV-2, SC) along the axis of the ovariole, showing that oogenesis of Artemia followed a polytrophic oocyte mode.At Stage I, the ovary was not fully developed, which was transparent with a small volume and lacked paired intact ovarioles (figure2b, I).The first phase oocytes (oogonia) had a high nucleo-cytoplasmic ratio, nearly round shape and highly homogeneous by HE staining (figure2b, I-2,3, dark arrow).The second phase oocytes (figure2b, II-3, dark arrow) occurred at Stage II with an irregular elliptical shape and larger size.The third phase oocytes (figure2b, II-3, dark arrow) were round or polygonal with prominent nucleoli, and the fourth phase oocytes (figure2b, II-4, dark arrow) had an irregular shape with increased cell volume.The oocyte maturation process was characterized by gradually increasing oocyte size and yolk granules, but a decreased nucleo-cytoplasmic ratio (figure2b, III-V).

Figure 1 .
Figure 1.Temporal patterns and morphology of ovarian development in A. parthenogenetica.dph, day post-hatching; Ana, anaphasis; E, eggs; I: Intestine; Meta, metaphasis; Oo, oocyte; dark arrows indicate ovariole bud; triangular white arrows indicate ovulatory hole; yellow dotted line indicate oocysts.I, II, III, IV and V indicate different stages of ovarian development, in sequence, as labelled.

Figure 2 .
Figure 2. (a) Schematic diagram of morphological changes in five stages of ovarian development of A. parthenogenetica.Red dot: cGSCs; Yellow circle: oocytes; Brown circle: eggs.(b) Cytological characteristics of oocytes accompanied by the ovarian development of A. parthenogenetica.Roman numerals I-V represent the five stages of ovarian development.Stage I: dark arrows indicate oogonia in the ovariole.Vec: vector section.Stage II: The white square dots in panel II-1 indicate, migration route of egg formation; Ant, anterior; Hemo, haemocoel; Lon, longitudinal section; Medi, medial surface; N, nucleus; Or, ovarioles; Out, outside.Stage III: FC, follicle cells (dark arrows); V, vitellogenin.Stage IV: SC, support cells; EC, egg chamber; Gr, germarium.White arrows indicate oocytes from different regions of the ovariole.Stage V: ES: eggshell; YG: yolk granules; white arrowhead in panel II-1: oviduct channel.

Figure 4 .Figure 5 .
Figure 4. (a) Differential expressed genes during the ovarian development of Artemia.(b) Volcanic plot and (c) scatter plot of KEGG enrichment of DEGs in the ovaries of Artemia at developmental Stage IV versus Stage II.

Figure 6 .
Figure 6.Protein structure of VgR in Artemia and phylogenetic analysis of VgRs in related species.(a) The structure domains of Ap-VgR protein of Artemia, including ligand-binding domain (LBD), EGF-precursor homology domain (EGFD) and transmembrane domain (TM).(b) Phylogenetic tree of VgRs in various oviparous species.

Figure 7 .Figure 8 .
Figure 7. Profiles of VgR expression in the process of ovarian development of Artemia.(a) Expression levels of Ap-VgR mRNA in intestine, cerebral ganglion and ovary of at five ovarian development stages (I-V).(b) Expression level of Ap-VgR mRNA on the first, third and fifth day post injection of dsEGFP and dsVgR.(c) Expression levels of VgA, VgB, Gbb and Dpp mRNA on the first day post dsRNA injection.Data are expressed as mean ± s.d.(n = 30).Different letters represent significant difference among the groups ( p < 0.01).Asterisks indicate significant differences between two groups (*p < 0.05, **p < 0.01).

Figure 9 .
Figure 9. Offspring number per individual of Artemia on the third and fifth days after the ovulation period (n = 30).Double asterisk indicates a significant difference between two groups ( p < 0.01).