Wolbachia infection in West Nile Virus vectors of northwest Iran

Mosquito-borne viral diseases are serious health problems in many countries. Various methods have been used for controlling the vectors of these diseases. Among symbiotic bacteria, the members of the genus Wolbachia are the most ubiquitous symbionts of arthropods and play key roles in their host biological characteristics with various effects on their hosts. The identification of these bacteria in Iranian mosquitoes is limited to a few studies. The current study was carried out to determine (1) the Wolbachia infection of probable arbovirus vectors (Aedes caspius, Culex pipiens, Culex theileri and Culiseta longiareolata), (2) the Wolbachia strain(s) infecting the mosquitoes, and (3) the geographical distribution of the Wolbachia strain(s) in the northwest of Iran. Eight species including Ae. caspius, Anopheles hyrcanus, An. maculipennis, Cx. hortensis, Cx. modestus, Cx. pipiens, Cx. theileri, and Cs. longiareolata were identified, amongst which Ae. caspius with 63.1% and An. hyrcanus with 0.3% were the most and the least abundant species, respectively. The results of semi-nested PCR using Wolbachia surface protein (wsp) fragment assays showed that Wolbachia infection was present in three out of the four above mentioned arboviral vector species (Aedes caspius, Culex pipiens, Culex theileri and Culiseta longiareolata), where the highest infection rate was seen in Cx. pipiens. The infection rates of mosquitoes with Wolbachia in the species of Cx. pipiens, Cs. longiareolata, Cx. theileri, and Ae. caspius were 96.9%, 11.5%, 5.2% and 0%, respectively.

     

Comparison of different trapping methods for surveillance of mosquito vectors of West Nile virus in Rhône Delta,France

Five trapping methods were compared for monitoring potential vectors of the West Nile virus in four areas in the Camargue Plain of France: carbon dioxide traps, bird‐baited traps, gravid traps, resting boxes, and human landing catches. A total of 73,721 specimens, representing 14 species, was trapped in 2006. Results showed significant differences in species and abundance between the type of traps. Many more specimens were collected using CO₂ traps than any other method, with an average of 212 specimens per night per trap (p<0.05). Culex pipiens was the most abundant species collected (36.8% of total with CO₂ traps), followed by Aedes caspius (22.7%), Anopheles hyrcanus (18.3%), Culex modestus (18.3%), and Aedes detritus (3.2%). Bird‐baited traps captured only eight specimens per night per trap on average, mainly Cx. pipiens (89.9%). The species collected and their abundance are influenced by the trap location, at ground or canopy level. Culex pipiens was twice as abundant in the canopy as on the ground, whereas it was the opposite for Ae. caspius, An. hyrcanus, and Ae. detritus. Culex modestus was equally abundant at both levels. Resting boxes and gravid traps were much less efficient, capturing around 0.3 specimens per night per trap. Results are discussed in relation to West Nile virus surveillance.     

Seasonal dynamics of mosquitoes (Diptera: Culicidae) in Osijek (Croatia) for the period 1995–2004

The ten year research (1995–2004) of adult mosquitoes in the area of Osijek by dry ice baited CDC traps has provided significant information on mosquito species, species prevalence, their dynamics and seasonal activity. A total of 207,136 adult mosquitoes comprising twenty species belonging to the following genera: Anopheles, Ochlerotatus, Aedes, Culex, Coquillettidia, Culiseta and Uranotaenia were collected. The seven most common species collected during this study were: Aedes vexans (75.6%), Ochlerotatus sticticus (13.3%), Culex pipiens complex (5.9%), Anopheles maculipennis complex (1.9%), Oc. cantans (0.9%), Oc. caspius (0.6%) and Oc. excrucians (0.6%). The temporal changes in the relative abundance of the twenty mosquito species in the Osijek area were reflected in three peaks: in May, July and September. Seasonal population dynamics differed between years and between mosquito species in relation to wetland, urban and forest habitats and was influenced by water level of the Drava and the Danube rivers.     

Flaviviral disease mosquito vector surveillance in Incheon Metropolitan City and the Hwaseong area,Gyeonggi‐Do,Republic of Korea,in 2015

Owing to global climate change, the global resurgence of vector‐borne infectious diseases and their potential to inflict widespread casualties among human populations has emerged as a pivotal burden on public health systems. In this study, the prevalence of flaviviral diseases transmitted by mosquitoes, and their target vector diversity, abundance, and distribution was investigated to enable the mapping of hotspots for these diseases. For the surveillance of the vector mosquitoes carrying flaviviruses during April to November 2015, female mosquitoes were collected to study whether they carried pathogens from abroad at seven locations in Incheon Metropolitan City (Incheon) as a typical urban area and Hwaseong‐si (= city, Hwaseong) of Gyeonggi‐do (= province) as a rural area. A total of 15 species belonging to seven genera (29,102 female mosquitoes) were collected with black‐light and BG‐Sentinel? traps at a collection rate of 260 per trap/night from whole collection locations. The most collected mosquito species in Incheon were Aedes vexans nipponii (species ratio (SR), 29.9%) and the Culex pipiens complex (SR, 28.8%), followed by Anopheles sinensis s.l. (SR, 27.9%) and Ochlerotatus koreicus (SR, 7.1%). From the results of viral RNA detection, five flaviviruses were found in 20,981 individuals (excluding An. sinensis; 696 pools) in the Cx. pipiens complex and Ae. vexans nipponii. Three Japanese encephalitis virus (JEV)‐positive pools were from the Cx. pipiens complex, a Chaoyang virus pool was found from Ae. vexans nipponii, and the remaining unidentified flavivirus pool was from Cx. pipiens. The three JEV‐positive pools were phylogenetically grouped as genotype V. The results of our study demonstrate that enhanced monitoring and long‐term surveillance of these vector viruses are of great public health importance.     

The mosquitoes of Finland: updated distributions and bionomics

Mosquitoes (Diptera: Culicidae) were collected in Finland between 2012 and 2018 to determine the species present and their distributions. In total, 52 466 specimens from 1031 collections formed the basis for the preparation of distribution maps for each of the 40 species that were collected. Anopheles maculipennis s.s., An. claviger, Aedes geminus and Ochlerotatus sticticus are confirmed on mainland Finland after previous records were uncertain or absent. Coquillettidia richiardii, Culiseta morsitans, Cs. ochroptera, Culex territans, Cx. torrentium, Oc. leucomelas, Oc. nigrinus, Oc. pullatus and Oc. punctodes occur more widely than previously reported. Three species, Ae. rossicus, Cs. subochrea and Oc. cyprius were not collected, although Ae. rossicus was subsequently found in Lapland by another researcher. No invasive species were collected. Ochlerotatus communis, an aggressive biter, was the most commonly encountered species. Larval collection data suggest that several species may have up to three generations per year in Finland, with Cx. torrentium and Cx. pipiens having at least two, and Oc. communis and Oc. punctor regularly found as larvae across the summer. These data, especially when coupled with historical records, are vital for monitoring species which have significant vector potential, particularly when faced with a warming climate.     

The mosquito fauna of the western region of Spain with emphasis on ecological factors and the characterization of Culex pipiens forms

This study updates the diversity, distribution, and seasonal trends of mosquitoes in a western region of Spain, assesses ecological determinants of Culex pipiens s.l., and determines form composition of Cx. pipiens s.s. populations. A total of 1,495 mosquitoes of 16 species was collected during 2012–2013, of which Cx. pipiens s.l. and Cx. theileri were the most abundant. Five new records for An. maculipennis s.s., Orthopodomyia pulcripalpis, Aedes (Ochlerotatus) punctor, Cx. europaeus, and Cx. modestus were found for this region. Cx. pipiens density varied across weather and habitat patterns, correlating positively with high temperatures and with a preference for urbanized areas and rural areas within a proximity of ovine farms. Moreover, molecular identification by CQ11FL was performed in 467 Cx. pipiens s.s., detecting both pipiens (66%) and molestus (8.4%) forms coexisting in different habitats (urban, peri‐urban and rural) aboveground with a high degree of hybridization (25.7%). The abundance of Cx. pipiens in urban areas and farms, with the presence of hybrids, may increase their capacity to act as bridge vectors for the transmission of arboviral infections. These data will be helpful for further implementation of entomological programs focused on risk assessment for arboviruses or other mosquito‐borne pathogens.     

A two-year survey on mosquitoes of Lebanon

A total of 6,500 mosquitoes were identified during a two-year survey (1999-2001) in Lebanon, and these belonged to twelve species: Culex pipiens, Cx. laticinctus, Cx. mimeticus, Cx. hortensis, Cx. judaicus, Aedes aegypti, Ae. cretinus, Ochlerotatus caspius, Oc. geniculatus, Oc. pulchritarsis, Culiseta longiareolata and Anopheles claviger. Culex pipiens was the most predominant species in Lebanon, collected indoors and outdoors. It was continuously abundant and active throughout the year. Culex judaicus was a small and rare mosquito and it is reported to occur for the first time in Lebanon. On the coastal areas, Ochlerotatus caspius was very common, and proved to be a complex of species as two forms were detected. One of the vectors of malaria, Anopheles claviger, was collected from May to September, from eight sites in Lebanon. Its breeding sites were restricted to fresh, cool, and clean water in pools and wells. Most of these breeding sites were in the populated Metn County where a few indigenous cases of malaria were reported from 1997-2000. This shows that the reported malaria cases were not imported, but caused by the bites of locally infected Anopheles females.     

Ecological Niche Modelling of Potential West Nile Virus Vector Mosquito Species and Their Geographical Association with Equine Epizootics in Italy

In Italy, West Nile virus (WNV) equine outbreaks have occurred annually since 2008. Characterizing WNV vector habitat requirements allows for the identification of areas at risk of viral amplification and transmission. Maxent-based ecological niche models were developed using literature records of 13 potential WNV Italian vector mosquito species to predict their habitat suitability range and to investigate possible geographical associations with WNV equine outbreak occurrence in Italy from 2008 to 2010. The contribution of different environmental variables to the niche models was also assessed. Suitable habitats for Culex pipiens, Aedes albopictus, and Anopheles maculipennis were widely distributed; Culex modestus, Ochlerotatus geniculatus, Ochlerotatus caspius, Coquillettidia richiardii, Aedes vexans, and Anopheles plumbeus were concentrated in north-central Italy; Aedes cinereus, Culex theileri, Ochlerotatus dorsalis, and Culiseta longiareolata were restricted to coastal/southern areas. Elevation, temperature, and precipitation variables showed the highest predictive power. Host population and landscape variables provided minor contributions. WNV equine outbreaks had a significantly higher probability to occur in habitats suitable for Cx. modestus and Cx. pipiens, providing circumstantial evidence that the potential distribution of these two species coincides geographically with the observed distribution of the disease in equines.     

Distribution and ecology of mosquitoes in a focus of dirofilariasis in northwestern Iran, with the first finding of filarial larvae in naturally infected local mosquitoes

A study of the distribution and ecology of mosquitoes (Diptera: Culicidae) in Ardebil Province of northwestern Iran, with special reference to the known dirofilariasis focus in Meshkinshahr County, was carried out in July‐August 2005 and April‐October 2006. In total, 17 533 adult mosquitoes and 3090 third‐ and fourth‐instar larvae were collected on 14 occasions and identified using morphological characters and DNA sequence data. Twenty species belonging to seven genera were found: Anopheles claviger (Meigen), An. hyrcanus (Pallas), An. maculipennis Meigen, An. pseudopictus Grassi*, An. sacharovi Favre, An. superpictus Grassi, Aedes vexans (Meigen)*, Coquillettidia richiardii (Ficalbi)*, Culex hortensis Ficalbi, Cx. modestus Ficalbi, Cx. pipiens Linnaeus, Cx. theileri Theobald, Cx. torrentium Martini*, Cx. tritaeniorhynchus Giles, Culiseta longiareolata (Macquart), Cs. annulata (Schrank)*, Cs. subochrea (Edwards), Ochlerotatus caspius (Pallas) s.l.* (= Aedes caspius sensu auctorum), Oc. geniculatus (Olivier)* (= Aedes geniculatus sensu auctorum) and Uranotaenia unguiculata Edwards (asterisks indicate new occurrence records for the province). The most prevalent species in adult catches were An. maculipennis (52%), Cx. theileri (45%) and Cx. hortensis (1%); the most prevalent species caught as larvae were Cx. theileri (27%), Cx. hortensis (21%) and An. maculipennis (19%). Anopheles maculipennis, Cx. pipiens and Cx. theileri were most widely distributed in the province. The occurrence of Cx. torrentium in Iran is verified based on differential characters of fourth‐instar larvae. Anopheles maculipennis and An. sacharovi of the Maculipennis Group were identified from their diagnostic ITS2 sequences. For the first time, cytochrome c oxidase I (COI) sequences were obtained from Iranian specimens of An. hyrcanus, An. pseudopictus, Cx. theileri and Oc. caspiuss.l. Culex theileri and An. maculipennis were found naturally infected with third‐stage (infective) larvae of Dirofilaria immitis (Leidy) and Setaria labiatopapillosa (Alessandrini) (Spirurida: Onchocercidae), respectively, for the first time in Iran.     

Laboratory Evaluation of Controlled-release Repellent Treated Pulp Fabric on Human Volunteers Against Mosquito Vectors

Three controlled‐release personal‐use pulp fabric impregnated insect repellent formulations of 5% N, N‐diethyl‐3‐methylbenzamide (DEET), and 10% and 15% neem oils were evaluated in an environmental chamber on volunteers for their repellent efficacy against three mosquito species, Culex pipiens pallens, Aedes aegypti and Ochlerotatus togoi. The 5% DEET formulation showed significant repellency in pulp fabric (5 mm in width) against Culex pipiens pallens and Aedes aegypti (P < 0.05), providing an average repellency of 88.0%, 66.3%, and 46.8% of Cx. pipiens pallens, Ae. Aegypti and O. togoi bites, respectively, during the 6 hours of exposure period. Against night‐biting mosquitoes Cx. pipiens pallens, the DEET formulation provided mostly complete protection for at least 4 hours after the application. In pulp fabric of 10 mm in width, the 5% deet formulation showed significantly the highest repellency among the repellents against O. togoi (P<0.05), providing an average repellency of 52.3% during the 6 hours of exposure period. However, the pulp fabrics treated with 10% and 15% neem oil were less effective than 5% DEET against three mosquito species. This study demonstrated the potential of 5% DEET as pulp fabric repellent against both day‐ and night‐biting mosquitoes.     

Effects of blood meal sources on the biological characteristics of Aedes aegypti and Culex pipiens (Diptera: Culicidae)

Mosquitoes transmit many diseases to humans and animals e.g., malaria, yellow fever, dengue, filariasis and encephalitis. The fundamental target of this search was to study the effect of three different blood meal sources (human; rabbit and pigeon) on some biological and behavioral properties of Aedes aegypti and Culex pipiens. The results have assured that the females of the mosquito Ae. aegypti that were fed on human blood meal has registered the highest feeding activity from feeding on the blood meal whereas the females of the other mosquito Cx. pipiens have shown the highest feeding activity after being fed on pigeons when compared with its feeding on other factors. The results have shown non-significant variation in the average time necessary to digest the blood meal on both mosquito species Ae. aegypti and Cx. pipiens that were fed on vertebrate hosts under laboratory conditions. Furthermore, results assured that the difference in blood meal sources has yielded distinct variation in the reproductive capacity and efficiency of both female mosquitoes under investigation where both species Ae. aegypti and Cx. pipiens already fed on human blood meal have yielded a pronounced distinctive increase in egg production (oviposition) when compared with females that were fed on pigeon or rabbit blood meal respectively. Moreover, feeding of the female mosquitoes under lab conditions on different blood meal sources did not affect the level of the hatching eggs that were laid by both mosquito females.     

Mosquitoes (Diptera: Culicidae) of the Lower Dyje River Basin (Podyjí) at the Czech-Austrian border

During 2009–2011, mosquitoes were captured in CDC miniature light traps using CO₂ (dry ice) at six sites in the Lower Dyje River Basin (Czech Republic). Other methods of capture — sweeping from vegetation and collection of larvae and pupae from ponds — were also used for more precise diagnostics. Thirty mosquito species of six genera were confirmed. A total of 415,218 females were captured. Most frequently found were the outbreak species Aedes vexans (56.52% of all mosquitoes collected) and Ae. sticticus (16.40%). Among other flood species, Ae. rossicus (5.17%), Ae. cantans and Ae. annulipes (2.44% of all females collected), and Ae. cinereus s. l. (1.11%) were especially abundant. Females of Ae. cataphylla were captured in spring (0.31%) and Ae. intrudens was numerous only at one site. Among the other species, Culex pipiens s. l. (6.61%) and Cx. modestus (8.87%) were abundant. Anopheles maculipennis s. l. (1.01%), An. claviger (0.43%), An. plumbeus (0.08%), An. hyrcanus (0.08%), Coquillettidia richiardii (0.52%) and Culiseta annulata (0.18%) were also detected. Sparsely occurring were Ae. excrucians, Ae. flavescens, Ae. caspius and Ae. geniculatus. Captured only very sporadically were Ae. communis, Ae. leucomelas, Ae. dorsalis, Ae. rusticus, Cx. martinii, Cx. territans, Cs. morsitans and Uranotaenia unguiculata.     

Experimental infection of Aedes (Stegomyia) albopictus and Culex pipiens mosquitoes with Bluetongue virus

Bluetongue disease (BT), caused by Bluetongue virus (BTV), infects wild and domestic ruminants, causing severe economic damage in the cattle and sheep industry. Proven vectors of BTV are biting midges belonging to the Culicoides genus, but other arthropods are considered potential vectors, such as ticks, mosquitoes, wingless flies, and sand flies. The present study represents the first attempt to evaluate the vectorial capacity of Culex pipiens and Aedes albopictus for BTV. Mosquitoes were artificially fed with blood containing BTV serotype 1. Infection, dissemination and transmission rates were evaluated at 0, 3, 7, 14 and 21 days after an infected blood meal. Viral RNA was only detected up to 3 days post infection in the bodies of both species. This study indicates that the two Italian populations of Cx. pipiens and Ae. albopictus are not susceptible to BTV infection.     

Evaluation of species composition and seasonal dynamics of mosquito larvae in the Košice Basin during 2010 and 2011

In six sites in the Ko?ice Basin we collected 17,520 larvae of 15 mosquito species during the seasons (April–August) of 2010 and 2011. They were: Aedes vexans (Meigen, 1830), Ae. cinereus (Meigen, 1818) [or Ae. rossicus (Dolbeskin, Gorickaja & Mitrofanova, 1930], Ochlerotatus geniculatus (Olivier, 1791), Oc. refiki (Medschid, 1928), Oc. rusticus (Rossi, 1790), Oc. sticticus (Meigen, 1838), Oc. punctor (Kirby, 1837), Oc. cataphylla (Dyar, 1916), Oc. cantans (Meigen, 1818)[or Oc. annulipes (Meigen, 1830)], Oc. communis (De Geer, 1776), Oc. flavescens (Müller, 1764), Oc. leucomelas (Meigen, 1804), Culiseta annulata (Schrank, 1776), Culex pipiens (L., 1758) [or Cx. torrentium (Martini, 1925)] and Anopheles maculipennis s.l. The objective of the present research was to identify the mosquito larvae species diversity and compare their distribution and density in urban and suburban localities of the monitored territory.     

Mosquitoes (Diptera,Culicidae) of the Morava River floodplain,Slovakia

Research on the Morava River floodplain mosquito fauna was carried out from April to October in 2009 and 2010. Altogether, 5864 adults were collected and identified from 12 selected sites. The presence of 28 mosquito species belonging to 6 genera were confirmed in the study area. The floodplain of the Morava River is known for its frequent inundation every year. The floods are quite irregular and often followed by the mass production of mosquitoes. The most abundant species were Aedes vexans (41.4%), Ae. cinereus (7.5%), Ae. rossicus (16.7%), Ochlerotatus sticticus (20.5%), and Culex pipiens (3.1%). Ae. vexans and Oc. sticticus are typical outbreak species for the Morava River floodplain. The years 2009 and 2010 differed in the number of floods, which influenced the mosquito faunal composition and abundance during the year.     

Evidence of vertical transmission of West Nile virus in field‐collected mosquitoes

Male and nulliparous female mosquitoes were surveyed for evidence of vertical WNV infection in East Baton Rouge Parish, Louisiana. Adult male mosquitoes collected by trapping and aspiration, and adult male and nulliparous female mosquitoes reared from field‐collected larvae were tested. Adult male Culex spp., female Aedes albopictus (Skuse), and female Culex quinquifasciatus Say mosquitoes that were collected as larvae were test‐positive for WNV RNA. Infectious WNV was detected using virus isolation in field‐collected male Aedes triseriatus Say and Culex salinarius Coquillett; these data represent the first field evidence of vertical transmission of WNV in Ae. triseriatus and Cx. salinarius.     

Mosquito community composition and abundance at contrasting sites in northern South Africa, 2014–2017

Most data on species associations and vector potential of mosquitoes in relation to arboviral infections in South Africa date back from the 1940s to late 1990s. Contextual information crucial for disease risk management and control, such as the sampling effort, diversity, abundance, and distribution of mosquitoes in large parts of South Africa still remains limited. Adult mosquitoes were collected routinely from two horse farms in Gauteng Province; two wildlife reserves in Limpopo Province, at Orpen Gate in Kruger National Park (KNP) and Mnisi Area in Mpumalanga Province between 2014–2017, using carbon dioxide‐baited light and tent traps. Mosquito diversity and richness are greater in untransformed natural and mixed rural settings. In untransformed wilderness areas, the most dominant species were Culex poicilipes, Anopheles coustani, and Aedes mcintoshi, while in mixed rural settings such as the Mnisi area, the two most abundant species were Cx. poicilipes and Mansonia uniformis. However, in peri‐urban areas, Cx. theileri, Cx. univittatus, and Cx. pipiens sensu lato were the most dominant. Aedes aegypti, Ae. mcintoshi, Ae. metallicus, Ae. vittatus, Cx. pipiens s.l., Cx. theileri, and Cx. univittatus had the widest geographical distribution in northern South Africa. Also collected were Anopheles arabiensis and An. vaneedeni, both known malaria vectors in South Africa. Arbovirus surveillance and vector control programs should be augmented in mixed rural and peri‐urban areas where the risk for mosquito‐borne disease transmission to humans and domestic stock is greater.     

Successional mosquito dynamics in surrogate treehole and ground‐container habitats in the northeastern United States: Where does Aedes albopictus fit in?

This study assessed the risk of larval displacement of the eastern treehole mosquito, Aedes triseriatus, and the northern house mosquito, Culex pipiens, by Aedes albopictus, the Asian tiger mosquito, during the establishment and successional stages of novel larval mosquito treehole and ground‐container habitats in the state of New Jersey, U.S.A. Culex pipiens and Culex restuans were the first mosquito species to colonize ground‐container habitats and were the dominant larval species throughout the study period, whereas Ae. albopictus was late to colonize ground habitats and accounted for less than 15% of weekly larval collections once established. Ae. albopictus had a much stronger community presence within treehole ovitraps; however, Ae. albopictus never reached the average larval densities of the expected primary colonizer, Ae. triseriatus. Throughout the study period, the weekly abundances of Ae. triseriatus and Ae. albopictus were positively correlated and there were no significant differences between the abundances of each species. The larval dominance of Ae. triseriatus appears to be enhanced by the presence of Toxorhynchites rutilus septentrionalis, a large predatory mosquito species. When Tx. rut. septentrionalis was present, mature larvae (3^rd–4^th instar) of Ae. albopictus were also present in only 16.7% of collections, whereas mature larvae of Ae. triseriatus were collected concurrently with Tx. rut. septentrionalis in 53.8% of collections. These data suggest that Ae. triseriatus is at a greater risk of displacement by Ae. albopictus than are Cx. pipiens and Cx. restuans.     

Novel spirochetes isolated from mosquitoes and black flies in the Czech Republic

During the years 1999–2002, a total of 4,898 individuals of 26 species of hematophagous insects (4,149 mosquitoes, 583 black flies, and 166 tabanid flies) was examined for the presence of spirochetes using dark‐field microscopy. There was an overall recovery of spirochetes from the midguts of Culicidae and Simuliidae of 23.5% and 11.4%, respectively. Spirochetes were not detected in Tabanidae. Seven spirochetal strains have been successfully recovered from mosquitoes and black flies: BR149 (Culex pipiens), BR151 (Cx. pipiens), BR173 (Cx. pipiens), BR177 (Cx. pipiens), BR193 (Aedes cinereus), BR208 (Cx. pipiens), and BR231 (Simulium noelleri). The strains have been adapted to laboratory conditions (BSK‐H Complete medium). Their preliminary determination based on 16S rRNA gene sequencing has shown that they differ from the Lyme disease spirochete Borrelia burgdorferi sensu lato as well as other members of the Order Spirochaetales indicating novel bacterial species in the Family Spirochaetaceae.