Abstract:To quantify the fish assemblage and its historical change, we conducted surveys in LongyangxiaReservoir in May, September, and December 2023 and analyzes the data against historical data. Nineteen speciesof fish were collected, belonging to 4 orders, 6 families, and 15 genera, among which Cyprinidae was thedominant family, accounting for 52.63% of the total species captured. The dominant species of fish inLongyangxia Reservoir were Carassius auratus, Oncorhynchus mykiss, Triplophysa scleroptera, Pseudorasboraparva, Hypomesus olidus, and Gymnocypris eckloni, and the dominant species of fish in the main stream of theYellow River were Gymnocypris eckloni, Schizopygopsis pylzovi, and Gymnodiptychus pachycheilus. The speciesdiversity index was highest in May and lower in September and December. ABC curve analysis showed that the Wvalue was greater than 0, the biomass dominance curve was above the abundance dominance curve, and thecommunity structure of Longyangxia Reservoir was relatively stable. Comparative analysis with data from 1981and 2005–2007 showed that, since the impoundment of Longyangxia Reservoir, indigenous fish decreased by twospecies and exotic fish increased by 10 species. Currently, Longyangxia Reservoir is dominated by fishes thatprefer quiet and slow water, are omnivorous, lower-middle, sedentary, exhibit sticky egg-laying, and the lowermouth. The ecological matrix of individual fish showed that the indigenous fish in Longyangxia Reservoir weremainly in the lower middle layer (R=0.556) and the lower mouth (R=0.889), which provided ecological nichevacancies for broadly adapted exotic fishes. The fish composition of Longyangxia Reservoir in 2023 showed asimilarity index of 0.33 (moderately dissimilar) with that of 1981, and a similarity index of 0.50 (moderatelydissimilar) with that of 2005–2007. The fish community structure changed significantly after the impoundment ofLongyangxia Reservoir in 1987. The analysis confirmed that the reservoir impoundment and the introduction ofexotic fishes were the main reasons for the changes in the fish community structure of the Longyangxia Reservoir.

Abstract:The source region of the Yellow River is located in the eastern region of the Qinghai-Tibet Plateau andthe ecological environment in the basin is fragile. The climate in this area is classified as plateau sub-cold zone,semi-humid and plateau temperate zone, semi-humid. The annual rainfall is approximately 300 mm, and theaverage annual water temperature is 3.4‒7.2 ℃. As the global climate is changing and human activities areincreasing, the ecological environment of the basin is confronting great pressure. The protection and restoration ofthe environment around the Yellow River has become the focus of human society. To explore the characteristics ofthe phytoplankton community structure and its relationship with environmental factors, two comprehensiveinvestigations were undertaken in the main stem and tributaries of the Yellow River in the spring and summer of2023, respectively. The total length of the survey site of the river was 1200 km. Phytoplankton samples werecollected from 33 survey stations along the source region of the Yellow River. The environmental parametersincluding water temperature, dissolved oxygen level, conductivity, turbidity, pH, chlorophyll a level, suspendedsolid levels, flow velocity, river width, transparency, altitude, latitude, and longitude were collected in the field.One liter of water was collected in a polytetrafluoride bottle and returned to the laboratory for analyses of waterquality indices, including total nitrogen and total phosphorus levels and the potassium permanganate index. Intotal, 235 species of phytoplankton belonging to seven genera were identified, with an average density andbiomass of 182.75×10⁴ cells/L and 55.35×10⁻³ mg/L, respectively. Diatoms were the dominant species, accountingfor 72.31% of all species. Seasonal changes in species composition and abundance existed, but were notsignificant. From the perspective of spatial dynamics, the phytoplankton species and their abundance in the mainstem proceeded to the tributaries. The number of phytoplankton species was highest in the downstream area, andthe average density and biomass of phytoplankton were highest in the middle reaches. The Shannon-Wienerdiversity index, Margalef’s richness index, and Pielou’s evenness index values were 2.87±0.66, 6.65±0.94, and0.77±0.17, respectively. Biodiversity analysis showed that the diversity index values of the phytoplanktoncommunity were higher in spring than in summer, and these seasonal differences were highly significant. Therewere nine dominant species of phytoplankton, mainly belonging to Bacillariophyta. The dominant species in bothspring and summer were Fragilaria intermedia, Fragilaria biceps, Synedra ulna, Diatoma moniliforme, and Cymbella turgidula. Correlation analyses between the phytoplankton community structure and environmentalfactors showed that the influencing factors of the phytoplankton community structure were different in spring andsummer in the source region of the Yellow River. The main factors affecting phytoplankton density and biomass inspring were altitude, river width, and flow velocity, while in summer the main factors were the potassiumpermanganate index value, chlorophyll a level, suspended solid level, and transparency. The results of this studyprovide an important foundation for scientific decision-making regarding basin ecological environment protectionand management in the Yellow River.

Abstract:To reveal the community structure characteristics of plankton in the Henan section of the Yellow River,three samplings were conducted at 15 sites in May 2023, October 2023, and February 2024. Qualitative andquantitative analyses of plankton in the samples were carried out. The analysis was conducted from five aspects:the species composition, density, biomass, dominant species, and biodiversity of plankton. A total of 134 species(genera) of phytoplankton belonging to 8 phyla, mainly Chlorophyta and Bacillariophyta, followed by Cyanophyta,were detected at the 15 sites. A total of 66 species (genera) of zooplankton belonging to four major groups weredetected, among which Rotifera had the largest number of species, followed by Protozoa. The abundance ofphytoplankton ranged from 6.02×10⁴ to 11.71×10⁶ cells/L, with an average of 10.92×10⁵ cells/L, and the biomasswas 0.06–5.83 mg/L, with an average of 0.66 mg/L. The abundance of zooplankton ranged from 3 to 2180 ind/L,with an average of 164.03 ind/L, and the biomass was 0.001–3.363 mg/L, with an average of 0.330 mg/L.One-way analysis of variance showed that there was a significant difference in the density of phytoplanktonbetween May and October (P＜0.05), but no significant difference between May and February (P ＞ 0.05). Therewere significant differences in the density of zooplankton between May and October and February (P＜0.05). Therewere significant differences in the biomass of zooplankton and phytoplankton between May and October andbetween May and February (P＜0.05). Chlorella sp., Ankistrodesmus spp., Chlamydomonas spp., Oocystis spp.,Melosira granulata, Synedra acus, Cyclotella spp., and Fragilaria spp. were the dominant species ofphytoplankton in different periods. Brachionus calyciflorus, Asplanchna girodi, Bosmina spp., Diaphanosoma spp.,Daphnia spp., Harpacticoida spp., and Nauplius spp. were the dominant species of zooplankton in differentperiods. The Shannon-Wiener index value of phytoplankton ranged from 0.66 to 4.38, with an average of 2.70. TheMargalef species richness index value was 1.32–4.68, with an average of 2.70. The Pielou species evenness indexvalue was 0.16–0.97, with an average of 0.63. The Shannon-Wiener index value of zooplankton ranged from 0.50to 3.02, with an average of 1.70. The Margalef species richness index value was 0.32–2.33, with an average of1.14. The Pielou species evenness index value was 0.39–1.00, with an average of 0.84. According to thebiodiversity index evaluation criteria, the recent water quality level of the Henan section of the Yellow River wasslightly polluted.

Abstract:The Baihe River and the Heihe River, which are both tributaries of the upper Yellow River in Sichuan Province, China, exhibit unique environmental and climatic conditions that support a diverse fish population. To investigate the species composition, community structure, and diversity of fish species in these rivers, this study incorporated historical data and field surveys that were conducted during the winter of 2022 (November), and in the spring (April), summer (July), and autumn (September) of 2023. A total of 2936 individuals from 11 species, spanning 8 genera, 2 families, and 1 order, were collected. Among these, the following four were endemic to the upper reaches of the Yellow River: Platypharodon extremus, Gymnocypris eckloni, Schizopygopsis pylzovi, and Chuanchia labiosa. Additionally, four species classified as second-class protected fish in China, namely, Triplophysa siluroides, Gymnodiptychus pachycheilus, P. extremus, and C. labiosa, and one provincial protected species, Triplophysa papptnhtimi, were detected. Two exotic species, Cyprinus carpio and Carassius auratus, represented 36.36%, 36.36%, 9.09%, and 18.18% of the total species identified in November 2022, April 2023, July 2023, and September 2023, respectively. Among the collected species, seven belonged to the Cyprinidae family and four to the Cobitidae family, accounting for 63.64% and 36.36% of the total number of species, respectively. The index of relative importance value indicated that the dominant species in the Baihe River were G. eckloni and Triplophysa scleroptera, while the dominant species in the Heihe River were G. eckloni and G. pachycheilus. The Shannon-Wiener diversity index value ranged from 1.764 to 2.364, the Margalef richness index value ranged from 1.364 to 1.798, and the Pielou evenness index value ranged from 0.7854 to 0.8747. Notably, the fish biodiversity index peaked in autumn. The abundance/biomass comparison curve indicated a stable fish community structure in both the Baihe River and Heihe River, dominated by large individual species with minimal external disturbances. Cluster and non-metric multidimensional scaling analysis revealed that the fish communities in the Baihe River and Heihe River could be classified into three distinct groups. Furthermore, when compared with historical data, there was a notable decrease in fish diversity in the Baihe River and Heihe River, accounting for 72.73% and 66.67% of the historical total number of species, respectively. The upper reaches of the Yellow River exhibited a high proportion of unique and protected fish species, characteristic of the typical Qinghai-Tibet (Central Asian) Plateau fish fauna. This study provides valuable data on fish community dynamics in the main tributaries of the Yellow River in Sichuan and offers recommendations for the protection of unique fish species in the upper reaches of the Yellow River.

Abstract:The Yellow River Delta is a typical representative of a newborn estuarine wetland, and is also an important ecological function area in both the Bohai Sea and the Yellow River Basin. Studying the shellfish community structure in the intertidal zone of the Yellow River Delta is of guiding significance for assessing the ecological status of the Yellow River Delta. In this study, the benthic shellfish resources of eight sampling sections in the intertidal area of the Yellow River Delta were systematically investigated to identify the seasonal variation in shellfish diversity and community structure characteristics in the Yellow River Delta. The investigations were conducted during astronomical tide in four seasons, namely, summer in August 2022, autumn in October 2022, winter in February 2023, and spring in May 2023. Twenty-two shellfish species were identified and the species quantities and densities, the dominant species, and the dominant degree varied greatly among both seasons and sampling sections. Most species (16) and the highest density of 1203.96 ind/m² were observed in summer. The dominant species were Umbonium thomasi, Potamocorbula laevis, Moevella iridescens, and Mactra quadrangularis, with P. laevis being dominant in all seasons. The Shannon-Wiener diversity, Margalef richness index, and Pielou evenness index values were highest in spring. U. thomasi, P. laevis, Mactra quadrangularis, and Meretrix petechialis were the four species with the largest contributions to inter-community dissimilarity among sampling sections in all four seasons. The results show that the benthic shellfish resource in the intertidal area of the Yellow River Delta was largest in summer, but the community diversity was highest in spring. Our results provide basic data and a theoretical foundation for the protection, development, and utilization of wild shellfish resources in the Yellow River Delta.

Abstract:The food composition of Gymnocypris eckloni, an indigenous fish species of Eling Lake at the source of the Yellow River, can lay a foundation for its biology. In the present study, feeding intensity, food composition, feeding habits, and their changes with the growth of Gymnocypris eckloni collected in June 2023 from Eling Lake were studied and analyzed by using microscopic analysis and DNA metabarcoding. A total of 7 taxa and 26 primary dietary groups were identified from the intestinal contents of 22 Gymnocypris eckloni using microscopic analysis. Metabarcoding technology detected 12 phyla and 43 genera of bait organisms. The food diversity index value and food species varied with the increase in size of Gymnocypris eckloni, and the dominant dietary group was Cnidaria. The diversity of bait organisms in the intestinal contents was higher for large Gymnocypris eckloni individuals than for medium-sized and small-sized Gymnocypris eckloni individuals. The present study, combining microscopic analysis and DNA metabarcoding, has greatly enhanced the ability to characterize the dietary habits of the Gymnocypris eckloni. This study provides important basic data and information for conducting artificial culture and determining the nutritional requirements and bait feeding and breeding characteristics of Gymnocypris eckloni.

Abstract:Lipids are important constituents and energy storage substances in fish, and their main role is oxidative energy supply. Excessive lipid levels in the feed affect the normal physiological metabolism of fish. KLF15 is an important regulator and effector of intra- and inter-organ metabolic processes, playing a key role in promoting fat synthesis and storage. TWIST2 is a conserved basic helix-loop-helix (b-HLH) transcription factor that serves as a molecular switch to activate or repress target genes through direct or indirect mechanisms, regulating pathways involved in inflammation, energy homeostasis, and metabolic disorders. The expression patterns of KLF15 with TWIST2 play important roles in many aquatic animals. This study aimed to investigate the role of the KLF15-TWIS2 signaling pathway in lipid metabolism in zebrafish (Danio rerio). Zebrafish larvae were randomly divided into a normal-fat diet (NFD) group and a high-fat diet (HFD) group 5 days after fertilization. The NFD group was fed a basal diet and the HFD group was fed an HFD. After 7 consecutive days of feeding, zebrafish litters from the NFD and HFD groups were homogenized in an ice bath to collect samples, centrifuged to extract the supernatant and measured to analyse changes in lipid metabolism-related indexes and the expression characteristics of the KLF15-TWIST2 signaling pathway. Physiological indices and antioxidant enzyme activities as well as histological observations were used to determine whether an HFD caused lipid metabolism disorders. The expression levels of KLF15, TWIST2, and genes related to lipid metabolism (SREBP, FASN, ACC1, DGAT2, CEBPα, CPT1, ATGL, LPL, SCD, and HSL) were detected by fluorescence quantitative PCR and their correlations were analyzed. An HFD caused a significant (P＜0.01) increase in total cholesterol, triglyceride, and low-density lipoprotein-cholesterol content in zebrafish. A significant (P＜0.05) increase in superoxide dismutase activity and a significant (P＜0.001) decrease in glutathione peroxidase activity were observed in zebrafish in the HFD group compared to those in the NFD group, and a large accumulation of lipids was observed. Intake of an HFD significantly increased the expression level of KLF15 (P＜0.001), but significantly decreased the expression level of TWIST2 (P＜0.05). The expression levels of lipid-synthesis-related genes, SREBP, FASN, ACC1, DGAT2, and CEBPα were significantly increased (P＜0.05), while the expression level of the lipolysis gene ATGL was significantly decreased (P＜0.05). Meanwhile, fatty acid oxidation genes PPARα, CPT1, LPL, LXPα showed a significantly higher expression level trend (P＜0.05). The expression levels of members of the KLF15-TWIST2 signaling pathway were significantly correlated with the expression levels of SREBP, FASN, ACC1, ATGL, DGAT2, CEBPα, PPARα, and LPL. These results suggested that an HFD led to excessive lipid accumulation and tissue damage and lipid metabolism disorders in zebrafish, and that the KLF15-TWIST2 signaling pathway may regulate lipid metabolism through its involvement in fatty acid synthesis, transport, β-oxidation, catabolism and lipoprotein synthesis. The present study explored the role of the KLF15-TWIST2 signaling pathway in lipid metabolism in fish, which is of great significance for understanding the health of fish.

Abstract:The increase in feed costs has significantly impacted the sustainable development of the Chinese abalone industry. The halophyte sea purslane (Sesuvium portulacastrum) is nutritionally valuable and shows potential as a novel bait source for abalone. We analyzed the nutrient composition of blanched and salted sea purslane to assess the value of sea purslane as abalone feed. Subsequently, blanched and salted sea purslane were separately used as the sole feed for Pacific abalone (＜em＞Haliotis discus hannai＜/em＞) with the same specifications in each group (shell length: 5.161±0.315 cm, body weight: 16.90±3.07 g). The red alga Gracilaria lemaneiformis served as the control feed. The abalone were feed once every 4 days over an 80-day culture cycle, with sufficient feed dispensed each time to ensure the provision of adequate feed and the generation of leftovers. There were four main findings. First, moisture, energy, crude protein (dry basis), crude fat, carbohydrate, and ash contents were (89.08± 0.01)%, (103.44±0.15) kJ/100 g, (12.49±0.28)%, (2.93±0.06)%, (20.34±0.28)%, and (29.10±0.08)%, respectively, for blanched sea purslane and (59.37±0.04)%, (139.38±0.79) kJ/100 g, (4.16±0.08)%, (1.25±0.01)%, (5.74±0.18)%, and (72.80±0.05)%, respectively, for salted sea purslane. Both types of sea purslane feed contained 18 amino acids, indicating a comprehensive amino acid profile that was relatively close to that of Pacific abalone muscle in terms of the presence of 11 important amino acids. Second, the two types of sea purslane feed significantly influenced the feed intake (FI) and feed conversion ratio (FCR) of Pacific abalone (P＜0.05). The FI and FCR were highest in abalone that fed on salted sea purslane and lowest in abalone that fed on G. lemaneiformis. Neither type of sea purslane feed significantly influenced the survival rate, average daily shell length gain, average daily body weight gain, specific growth rate of shell length, specific growth rate of body weight, condition factor, or meat yield of Pacific abalone (P˃0.05). Third, both types of sea purslane feed significantly increased the ash content and reduced the moisture, collagen, and total amino acid contents of Pacific abalone muscle (P＜0.05); however, they did not significantly influence the crude fat content, essential amino acids/total amino acids ratio, first limiting amino acid content(P˃0.05). The sea purslane feed could increase the linoleic acid and α-linolenic contents of Pacific abalone muscle. Fourth, there were no significant differences in the influence of the two types of sea purslane feed on the springiness and resilience of Pacific abalone muscle tissue (P˃0.05). However, blanched sea purslane significantly reduced the hardness and chewiness of the muscle tissue (P＜0.05), while salted sea purslane significantly increased the hardness of the muscle tissue (P＜0.05). In conclusion, both types of sea purslane feed can replace G. lemaneiformis as feed for Pacific abalone within an 80-day culture cycle. Pacific abalone fed with sea purslane had good production performance and nutritional value, with salted purslane contributing to improved abalone muscle texture.

Abstract:To investigate the variation in gut microbial diversity with food composition in Coilia nasus, 16S rRNA high-throughput sequencing technology was used to analyze the gut microbiota diversity of three different body length groups (L1: ≤140 mm, L2: 141–200 mm, L3: ≥201 mm) of C. nasus in the Yangtze River estuary. The analyses showed that copepods and nematodes were the main baiting taxa for C. nasus of the different length groups, and the clustering results showed that L1 was clustered into a single group, while L2 and L3 were clustered into a single group. A total of 726 genera and 378 families of microorganisms belonging to 30 phyla were identified in the intestinal samples. At the phylum classification level, the dominant groups of microorganisms in the different groups of C. nasus were similar, and were mainly composed of Actinobacteriota and Proteobacteria. Planctomycetota was only found in the intestines of L3 group. Rhodococcus was the common dominant genus in the intestines of C. nasus of different body lengths, with the highest abundance in group L1 (82.0%) and the lowest in group L3 (27.0%). Photobacterium was present at a certain percentage in all three groups. Alpha diversity analysis revealed that the gut microbial diversity of C. nasus increased with body length. Clustering analysis using the Bray-Curtis distance algorithm revealed that the clustering results of gut microorganisms among different groups were consistent with the clustering results of the stomach contents analysis. L1 was clustered into one group alone, and L2 and L3 were clustered into one group, which indicated that the composition of the food was closely related to that of the intestinal flora. Principal coordinates analysis showed that the L1 samples were separated from those of the other two groups, while the L2 and L3 samples were clustered together, which indicated that there was a difference in intestinal flora between the three groups. This study revealed that the food composition, feeding habits, and living environment of C. nasus were closely related to the composition of the intestinal microflora, which may provide a scientific basis for the conservation and rational development of C. nasus resources.

Abstract:Prorocentrum donghaiense is one of the main red tide organisms in China’s coastal waters. It is characterized by frequent outbreaks, a large area of influence, and serious harm. The cell cycle is an important biological process that regulates cell division. It can be affected by environmental factors, resulting in changes in the growth rate of phytoplankton. Therefore, it is valuable to investigate the differences in the cell cycle progression of P. donghaiense with different proliferation rates to help understanding its proliferation characteristics. The cell cycle pattern of P. donghaiense has been preliminary explored. Genes and proteins related to the cell cycle have been isolated and identified using omics approaches. However, there is a lack of research on the characteristics of the cell cycle with different proliferation rates, as well as the molecular mechanism. Our previous studies demonstrated that the growth rate of P. donghaiense varies under different environmental conditions. To compare the cell cycle progression of P. donghaiense with different proliferation rates and understand its molecular mechanism, we selected two experimental groups with slower (group 3) and faster (group 16) proliferation rates, and analyzed them using flow cytometry and RNA sequencing technology. The results showed that group 3 cells had a shorter S phase and were partially blocked in G2/M phase. A total of 149219 unigenes were obtained, of which 6081 were annotated in the NR, GO, KEGG, eggNOG, Swiss-Prot, and Pfam databases. A total of 114358 unigenes were classified into three terms, namely, cellular components, molecular functions, and bioprocesses, with 57 categories in the GO database. The 30554 unigenes annotated in the eggNOG database were classified into 25 categories. GO classification and pathway enrichment results of differentially expressed genes (DEGs) between the two experimental groups showed that the most important DEGs were related to microtubules and the cytoskeleton. We hypothesized that the differences in cell cycle progression between the two experimental groups were caused by differences in the dynamic changes of microtubules. Among the annotated unigenes, 49 encoding cyclins, 74 encoding cyclin-dependent kinases (CDKs), and 26 encoding cell division cycle (CDC) proteins were related to cell cycle control. Ten of these comprising one CYCA, one CYCB, one CYCU, one CDK1, four CDK2, and one Cdc48 genes were DEGs. The cyclinA-CDK2 and CDK1-cyclinB complexes participate in the cellular G2/M phase transition. Cells need to retain sufficient levels of CDK activity to maintain the transcription of G2-specific genes and retain their competence to recover from G2 arrest. Since cells in group 3 were partially arrested in G2/M, the transcriptional levels of CDKs, cylinA, and cyclinB were higher in group 3 than in group 16. As Cdc48 is involved in cellular DNA replication, the transcription level of Cdc48 was significantly higher in group 16 than in group 3, which explained why the S phase duration was longer in group 16 than in group 3. In summary, the S phase duration and whether the G2/M phase transition is blocked may decide the rate of P. donghaiense cell proliferation, and one CYCA, one CYCB, one CYCU, two CDK1, four CDK2, and one Cdc48 genes identified from the transcriptome analysis played important roles in cell cycle regulation.

Abstract:Eriocheir sinensis is an important economic species in China. It is widely distributed in the Yellow River, Yangtze River, and other river basins in China. It exhibits distinct feeding migrations and reproductive migrations. However, since the implementation of a 10-year fishing ban in the Yangtze River, it has been difficult to obtain fishery data. Therefore, a new method for monitoring E. sinensis resources under the background of the fishing ban in the Yangtze River needs to be established to monitor megalopa resources during the flood season. In this study, eDNA technology was used to monitor the abundance of E. sinensis megalopae, which helped reveal the dynamic changes in the population size of megalopae in the Yangtze River estuary and monitor the resource status of E. sinensis. By establishing an indoor quantitative curve and collecting eDNA samples from the Yangtze River Estuary, combined with megalopa resource survey data, the distribution of E. sinensis megalopa resources in the Yangtze River estuary was elucidated. The indoor quantitative curve of megalopae in the four concentration experiments(with concentration gradients of 1 megalopa per 20 liters of water, 10 megalopae, 100 megalopae, and 1000 megalopae) within 72 hours had the best fit with a power function. After removing the larvae, the eDNA concentration in the water was negatively correlated with time, and the power function best represented the relationship between eDNA degradation and time, which was consistent with previous research results for other crustaceans. In June 2023, the average density of megalopae in the Beibayao waters was (23.03±55.10) ind/m³, and the eDNA concentration range was (9145.86±31147.36) copies/mL. There was a highly significant correlation between the density of megalopae and the standardized eDNA concentration, and the power function equation had the best fit. The density of megalopae monitored using a plankton net in the Tuanjiesha waters was (0.38±0.99) individuals/m³, and the eDNA concentration was (29808.3±95359.04) copies/mL. There was a time-lag effect between megalopae density and eDNA concentration in the Tuanjiesha waters, with changes in density preceding changes in eDNA concentration by 5 days. After the lag, the density of megalopae was correlated with the standardized eDNA concentration, and the best-fit equation was a linear equation. The main reason for the lageffect was that the migration speed of megalopae was faster than the water exchange speed; thus, the megalopae arrived at the Tuanjiesha waters before the eDNA they produced. This study demostrates that the current method of using eDNA technology to analyze E. sinensis megalopa resources is only applicable to waters close to the occurrence of the megalopae.