Albopictus Females Research Articles

The effects of antibiotics on the reproductive physiology targeting ovaries in the Asian tiger mosquito, Aedes albopictus

AbstractMosquitoes have adapted to various environmental conditions. Symbionts with mosquitoes impact this adaptation in different environments. In the field, mosquitoes could get exposed to antibiotics during their developmental period, which could reduce or eliminate their symbiotic microbes. However, the side effects of the antibiotics on the ovary and reproductive physiology of the Asian tiger mosquito, Aedes albopictus remains unknown. In this study, we investigated the effects of tetracycline and combinations of rifampicin and tetracycline at environmentally acceptable levels on the reproductive physiology of ovaries in Ae. albopictus. Rifampicin and tetracycline in combination reduced the hatching rate and fertility of Ae. albopictus compared to the untreated control group. These antibiotics induced histopathological damage and reactive oxygen species production in the ovaries. The combination of antibiotics decreased the expression of surface protein of Wolbachia (WSP) in Ae. albopictus. Additionally, the expression of Toll like receptor 2 (TLR2) and Myd88 were triggered by the combinations. The findings demonstrate the detrimental effects of antibiotics, particularly combinations of rifampicin and tetracycline, on the reproductive capacity of Ae. albopictus females.

Reproductive interference between Aedes albopictus and Aedes flavopictus at a place of their origin.

Aedes (Stegomyia) albopictus and Aedes (Stegomyia) flavopictus are related species that have overlapping distributions from southern to central Japan. To understand how they interact, we studied reproductive interference between them, particularly focusing on the body size difference between the mating pair. Here, we examined the effects of conspecific, heterospecific and double mating (i.e. heterospecific mating followed by conspecific mating) on copulation duration, egg production and hatchability of eggs using mosquitoes that varied in body size. Females mated only with heterospecific males produced few viable eggs, indicating that post-mating isolation is almost complete. When mated with heterospecific males before conspecific mating, the production of viable eggs was lower than when mated only with conspecific males, revealing the occurrence of reproductive interference. The degree of reproductive interference was larger in Ae. flavopictus than in Ae. albopictus when the female size was small but did not differ between them when the female size was large. Aedes albopictus females appear to be able to distinguish Ae. flavopictus males from conspecific males and larger females are more successful in the rejection of heterospecific males. On the other hand, Ae. flavopictus were not able to discriminate between conspecific and heterospecific males.

Salvia Spp. Essential Oils against the Arboviruses Vector Aedes albopictus (Diptera: Culicidae): Bioactivity, Composition, and Sensorial Profile-Stage 1.

Mosquito-borne arboviruses diseases cause a substantial public health burden within their expanding range. To date, their control relies on synthetic insecticides and repellents aimed to control the competent mosquito vectors. However, their use is hampered by their high economic, environmental, and human health impacts. Natural products may represent a valid eco-friendly alternative to chemical pesticides to control mosquitoes, and mosquito-borne parasitic diseases. The aim of this work was to combine the chemical and sensorial profiles with the bioactivity data of Salvia spp. essential oils (EOs) to select the most suitable EO to be used as a repellent and insecticide against the invasive mosquito Aedes albopictus (Diptera: Culicidae), vector of pathogens and parasites, and to describe the EOs smell profile. To do this, the EOs of four Salvia species, namely S. dolomitica, S. dorisiana, S. sclarea, and S. somalensis were extracted, chemically analyzed and tested for their bioactivity as larvicides and repellents against Ae. albopictus. Then, the smell profiles of the EOs were described by a panel of assessors. The LC50 of the EOs ranged from 71.08 to 559.77 μL L−1 for S. dorisiana and S. sclarea, respectively. S. sclarea EO showed the highest repellence among the tested EOs against Ae. albopictus females (RD95 = 12.65 nL cm−2), while the most long-lasting, at the dose of 20 nL cm−2, was S. dorisiana (Complete Protection Time = 43.28 ± 3.43 min). S. sclarea EO showed the best smell profile, while S. dolomitica EO the worst one with a high number of off-flavors. Overall, all the EOs, with the exception of the S. dolomitica one, were indicated as suitable for “environmental protection”, while S. dorisiana and S. sclarea were indicated as suitable also for “Body care”.

Comparative Analysis of the Bacterial and Fungal Communities in the Gut and the Crop of Aedes albopictus Mosquitoes: A Preliminary Study.

The Asian tiger mosquito Aedes albopictus is a major pathogen vector and one of the world’s most invasive species. In recent years, the study of mosquito-associated microbiota has received growing interest for reducing transmission of mosquito-borne pathogens. Most of studies on mosquito microbiota mainly focused on the gut bacteria. However, microorganisms can also colonize other organs and are not restricted to bacteria. In mosquitoes, the crop is the primary storage organ for sugars from the nectar feeding before it is transferred into the midgut for digestion. No study has yet investigated whether this organ can harbor microorganisms in Ae. albopictus. By using high-throughput sequencing, this study is the first to describe the microbiota including both bacteria and fungi in sugar-fed Ae. albopictus males and females. The results showed the presence of diverse and rich bacterial and fungal communities in the crop of both sexes that did not strongly differ from the community composition and structure found in the gut. Altogether, our results provide a thorough description of the crop-associated microbiota in Ae. albopictus which can open new avenues for further studies on trophic interactions between the mosquito and its microbiota.

Oogenic development and gonotrophic cycle of Aedes aegypti and Aedes albopictus in laboratory.

To determine the time of oogenic development and the length of the gonotrophic cycle of Ae. aegypti and Ae. albopictus in laboratory. Bloodfed females of Ae. aegypti and Ae. albopictus were dissected every 4 h to determine the development status of the follicles according to the Christophers' stages. The minimum time of oocyte maturation in Ae. aegypti and Ae. albopictus was 64-82 h and 52-64 h post-feeding, respectively. We found that the gonotrophic cycle of Ae. aegypti (3.7-4.2 d) is longer than that of Ae. albopictus (3.2-3.7 d). The follicle length showed significant differences between species at Christophers' stages 2" and 5, whereas follicle amplitude was different between the two mosquitoes at stages 2", 3 and 4. The study provided new evidence on the reproductive strategies of Ae. aegypti and Ae. albopictus females that coexist in the Neotropical region of Mexico.

Arbovirus expansion: New species of culicids infected by the Chikungunya virus in an urban park of Brazil.

The Dengue, Zika and Chikungunya viruses have been spreading in tropical regions, causing epidemics with high morbidity rates and fatal cases. The aim of this study was to assess the diversity and abundance of culicid species and the presence of arboviruses in mosquitoes, at the epicenter of an epidemic outbreak that occurred in people living near an urban Atlantic Forest park. Mosquitoes were captured with a Shannon trap between 2 and 6 pm in seven months of 2019. The Chikungunya virus was investigated according to the protocol described by Lanciotti (2007). The most abundant species were Wyeomyia bourrouli (66.9%) and Aedes albopictus (23.9%). Also captured were Aedes fluviatilis (3.2%); Haemagogus leucocelaenus (2.2%); Aedes scapularis (2.2%); Aedes aegypti (1.6%); Aedes serratus and (0.3%) and Aedes taeniorhynchus (0.3%). The Chikungunya virus was identified in A. aegypti females; A. albopictus females and males; Aedes fluviatilis and Wy.bourrouli. The presence of the Chikungunya virus in the afore mentioned mosquitoes reinforces the hypothesis that arbovirus expansion is associated with the participation of other mosquito species in the transmission areas, primarily the Chikungunya virus in the study area. The data also demonstrate the need for permanent entomological surveillance and measures to preserve the area, in order to hinder its degradation, the adaptation of culicid species to new habitats and the formation of enzootic cycles of these viruses in the forest.

Differential Small RNA Responses against Co-Infecting Insect-Specific Viruses in Aedes albopictus Mosquitoes

The mosquito antiviral response has mainly been studied in the context of arthropod-borne virus (arbovirus) infection in female mosquitoes. However, in nature, both female and male mosquitoes are frequently infected with insect-specific viruses (ISVs). ISVs are capable of infecting the reproductive organs of both sexes and are primarily maintained by vertical transmission. Since the RNA interference (RNAi)-mediated antiviral response plays an important antiviral role in mosquitoes, ISVs constitute a relevant model to study sex-dependent antiviral responses. Using a naturally generated viral stock containing three distinct ISVs, Aedes flavivirus (AEFV), Menghai rhabdovirus (MERV), and Shinobi tetra virus (SHTV), we infected adult Aedes albopictus females and males and generated small RNA libraries from ovaries, testes, and the remainder of the body. Overall, both female and male mosquitoes showed unique small RNA profiles to each co-infecting ISV regardless of the sex or tissue tested. While all three ISVs generated virus-derived siRNAs, only MERV generated virus-derived piRNAs. We also studied the expression of PIWI genes in reproductive tissues and carcasses. In contrast to Piwi5-9, Piwi1-4 were abundantly expressed in ovaries and testes, suggesting that Piwi5-9 are involved in exogenous viral piRNA production. Together, our results show that ISV-infected Aedes albopictus produce viral small RNAs in a virus-specific manner and that male mosquitoes mount a similar small RNA-mediated antiviral response to that of females.

Transstadial metabolic priming mediated by larval nutrition in female Aedes albopictus mosquitoes

Mosquitoes are important vectors of human pathogens, which are transmitted by female mosquitoes via blood-feeding. Larval nutrition can have an important impact on the number of blood meals taken by adult females shortly after emergence, as nutritional deficiencies during the larval stage may result in pre-vitellogenic blood meals, which are not invested into egg development but into the endogenous nutrient reserves of the female. Here, we investigated the impact of nutrient deprivation during the larval stage on adult nutrient metabolism, longevity and blood-seeking behaviour in females of the invasive Asian tiger mosquito Aedes albopictus. We demonstrate that Ae. albopictus females are able to compensate for nutrient deprivation during the larval stage by increasing their development time until sufficient nutrients are acquired. Nonetheless, nutrient-poor larval conditions had a long-lasting priming effect on adult female metabolism, since these females accumulated lower nutrient reserves from carbohydrates and survived longer compared to females reared in nutrient-rich larval conditions. Moreover, nutrient and ATP levels of females from nutrient-poor larval conditions remained stable over a longer timespan without access to additional carbohydrates. This suggests differences in adult female metabolism in response to larval nutrition, with potential impact on the vectorial capacity of female mosquitoes.

BITING PERSISTENCE IN AEDES ALBOPICTUS (DIPTERA: CULICIDAE)

Mosquitoes body size infuences a number of important factors determinants in disease transmission. Generally, high nutrition during developing stages results in large adults. The interrupted feeding of Ae. albopictus spreads zoonotic viruses. Adult dry weight and total number of landings positively correlated with the larval nutrition represented by adult body size. Potentiality of Ae. albopictus to attack its hosts for blood meal required for egg development is an important factor in disease transmission. Although the biting persistence was described in the literature as the norm for mosquitoes, yet more study was needed. Measurement of the number of landings during a 60-min test for large and small femalessupplied different nutrients. Small females were significantly less persistent than larger ones. Supply of 10% sucrose solution or water ± 10% sucrose solution for 6 days significantly influenced biting persistence in large and small females. The pattern of persistence exhibited more increase in case of females fed 10% sucrose solution and 10% sucrose solution+ water for 6 days than others fed for 4 days or 2 days, either from the high or low larval diet. This support the hypothesis that Ae. albopictus females body size influences bitting persistence.

Rat volatiles as an attractant source for the Asian tiger mosquito, Aedes albopictus

Aedes albopictus is a vector of dengue, chikungunya, and dirofilariasis. Volatile compounds are crucial for mosquitoes to locate their hosts. This knowledge has allowed the identification of attractants derived from human odours for highly anthropophilic mosquito species. In this study, we used rats as a experimental model to identify potential attractants for host-seeking Ae. albopictus females. Porapak Q extracts from immature female rats were more attractive to Ae. albopictus females than those from mature and pregnant females, and males. Phenol, 4-methylphenol, 4-ethylphenol, and indole were identified compounds in male, immature, mature, and pregnant female extracts. There were quantitative differences in these compounds among the extracts that likely explain the discrepancy in their attractiveness. Ae. albopictus females were not attracted to the single compounds when was compared with the four-component blend. However, the binary blend of 4-methylphenol + 4-ethylphenol and the tertiary blend of 4-methylphenol + 4-ethylphenol + indole were as attractive as the four-component blend. In the field trials, BGS traps baited with the tertiary or quaternary blends caught more Ae. albopictus females and males than BGS traps without lures. This is the first laboratory and field study to identify compounds that mediate the attraction of Ae. albopictus to one of its hosts.

Bionomics and insecticide resistance of Aedes albopictus in Shandong, a high latitude and high-risk dengue transmission area in China

BackgroundDengue fever outbreaks tend to spread northward in China, and Jining is the northernmost region where local dengue fever cases have been detected. Therefore, it is important to investigate the density of Aedes albopictus and its resistance to deltamethrin.MethodsThe Breteau index (BI) and container index (CI) were calculated to assess the larval density of Ae. albopictus and human-baited double net trap (HDN) surveillance was performed in six subordinate counties (Rencheng, Yanzhou, Sishui, Liangshan, Zoucheng and Jiaxiang) of Jining City in 2017 and 2018. The resistance of Ae. albopictus adults to deltamethrin was evaluated using the World Health Organization (WHO) standard resistance bioassay. The mutations at Vgsc codons 1532 and 1534 were also analysed to determine the association between kdr mutations and phenotypic resistance in adult mosquitoes.ResultsThe average BI, CI and biting rate at Jining were 45.30, 16.02 and 1.97 (female /man/hour) in 2017 and 15.95, 7.86 and 0.59 f/m/h in 2018, respectively. In August 26, 2017, when the first dengue fever case was diagnosed, the BI at Qianli village in Jiaxiang County was 107.27. The application of prevention and control measures by the government sharply decreased the BI to a value of 4.95 in September 3, 2017. The mortality of field-collected Ae. albopictus females from Jiaxiang was 41.98%. I1532T, F1534L and F1534S mutations were found in domain III of the Vgsc gene. This study provides the first demonstration that both I1532T and F1534S mutations are positively correlated with the deltamethrin-resistant phenotype.ConclusionsMosquito density surveillance, resistance monitoring and risk assessment should be strengthened in areas at risk for dengue to ensure the sustainable control of Ae. albopictus and thus the prevention and control of dengue transmission.

Active dispersal of Aedes albopictus: a mark-release-recapture study using self-marking units

BackgroundUnderstanding the dispersal dynamics of invasive mosquito species is fundamental to improve vector surveillance and to target control efforts. Aedes albopictus has been deemed a poor flyer as its range of active dispersal is generally assumed to be limited to a few hundred metres, while laboratory studies suggest this mosquito could actually fly several kilometres. The discrepancy may be due to differences in the local environment or to the methodological approach. In Switzerland, Ae. albopictus has been present since 2003 and has since then expanded its range. While passive dispersal is a key driver, it remains unclear how far this mosquito spreads through active flight and what the age structure and size of the local population are, all important parameters for vector surveillance and control.MethodWe investigated the active dispersal, daily survival rate and population size of Ae. albopictus in mark-release-recapture studies in Coldrerio and Lugano, two areas of intensive control in Switzerland. To mark mosquitoes emerging from breeding sites, we used self-marking units with fluorescent pigment that have minimal impact on mosquito survival and behaviour. We recaptured the adult mosquitoes with BG-Sentinel traps within a radius of 1 km from the marking units over 22 consecutive days.ResultsWe found that 77.5% of the recaptured Ae. albopictus individuals flew further than 250 m, the limit that is usually deemed sufficient for vector containment. The average age of females and males was 8.6 days and 7.8 days in Coldrerio and Lugano, respectively, while the estimated mosquito population densities were 134 mosquitoes/ha in Coldrerio and 767 mosquitoes/ha in Lugano.ConclusionsSelf-marking units are an effective tool to mark wild mosquitoes. Using this approach, we found that mosquitoes survive long enough to potentially transmit arboviral disease in our study area and that host-seeking Ae. albopictus females may travel further than previously assumed for European mosquito populations. This finding has direct implications for vector control as emergency treatments around positive cases, as well as surveillance and control around detections of new infestations, might need to be extended beyond the usual recommended range of just a few hundred metres.

Parity and gonotrophic discordance of females of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in the city of São Paulo, SP, Brazil.

The objective of this study was to assess the parity, presence of blood in the stomach, and the gonotrophic discordance of females of Aedes aegypti and Aedes albopictus captured in two areas of the city of São Paulo. The captures were undertaken monthly, by aspiration, in the period from January, 2015 to August, 2017. All the females of the two species had their midguts and ovaries dissected to determine the presence of blood and the parity/stage of maturation. With regard to parity, 27% and 34% of the females of Ae. aegypti and Ae. albopictus, respectively, were parous or were in advanced stages of the development of their ovaries (33% and 27%, respectively). The larger part of the females of Ae. aegypti and Ae. albopictus contained blood in their stomachs (77% and 60%, respectively), beyond which 36% and 27% of the females of Ae. aegypti and Ae. albopictus, respectively, were in gonotrophic discordance. Our results indicate favorable conditions in the study areas because of the presence of parous females. Moreover, this frequent and multiple contact of Ae. aegypti and Ae. albopictus females with vertebrate hosts, such as humans, increases the possibility of transmitting the viruses they may be carrying.

Identification of human skin bacteria attractive to the Asian Tiger mosquito.

Aedes albopictus is a vector of arboviruses and filarial nematodes. Originating from Asia, this mosquito has rapidly expanded its geographical distribution and colonized areas across both temperate and tropical regions. Due to the increase in insecticide resistance, the use of environmentally friendly vector control methods is encouraged worldwide. Using methods based on semiochemicals in baited traps are promising for management of mosquito populations. Interestingly, human skin microbiota was shown to generate volatile compounds that attract the mosquito species Anopheles gambiae and Aedes aegypti. Here, we investigated the composition of skin bacteria from different volunteers and the attractive potential of individual isolates to nulliparous Ae. albopictus females. We showed that three out of 16 tested isolates were more attractive and two were more repulsive. We identified dodecenol as being preferentially produced by attractive isolates and 2-methyl-1-butanol (and to a lesser extent 3-methyl-1-butanol) as being overproduced by these isolates compared with the other ones. Those bacterial volatile organic compounds represent promising candidates but further studies are needed to evaluate their potential application for baited traps improvement.

Estimating Spatio-Temporal Dynamics of Aedes Albopictus Dispersal to Guide Control Interventions in Case of Exotic Arboviruses in Temperate Regions

The increasing number of exotic arbovirus cases imported in Europe and the 2017 chikungunya outbreak in central/southern Italy highlight the urgency of evidence-based outbreak management plans to predict, prevent or interrupt spreading of these arboviruses to non-endemic countries in temperate regions. We here present the results of three mark-release-recapture experiments conducted in a peri-urban area of North-East Italy to estimate the spatio-temporal dynamics of the dispersal of Aedes albopictus females looking for oviposition sites. The Flight Range of 90% of the mosquito population (FR90) was found to exceed 200 m, consistently with data obtained from a previous study conducted in a highly urbanised area in Rome (Central Italy). Modelling results showed that dispersal can be so rapid that insecticide spraying within a 200m-radius around a potential infected case leaves >10% probability that a potentially infected mosquito escapes the treatment, even if this is carried out after only 2–3 days since the importation of a viremic case. These data provide evidence in favour of an update of guidelines for the control of exotic autochthonous arbovirus transmission in temperate areas and highlight the need of effective surveillance approaches and rapid response to contain the risks associated to imported viremic cases.

Fat body-specific vitellogenin expression regulates host-seeking behaviour in the mosquito Aedes albopictus.

The high vector competence of mosquitoes is intrinsically linked to their reproductive strategy because females need a vertebrate blood meal to develop large batches of eggs. However, the molecular mechanisms and pathways regulating mosquito host-seeking behaviour are largely unknown. Here, we test whether host-seeking behaviour may be linked to the female’s energy reserves, with low energy levels triggering the search for a nutrient-rich blood meal. Our results demonstrate that sugar feeding delays host-seeking behaviour in the invasive tiger mosquito Aedes albopictus, but the levels of energy reserves do not correlate with changes in host-seeking behaviour. Using tissue-specific gene expression analyses, we show for the first time, to our knowledge, that sugar feeding alone induces a transient up-regulation of several vitellogenesis-related genes in the female fat body, resembling the transcriptional response after a blood meal. Specifically, high expression levels of a vitellogenin gene (Vg-2) correlated with the lowest host-seeking activity of sugar-fed females. Knocking down the Vg-2 gene via RNA interference (RNAi) restored host-seeking behaviour in these females, firmly establishing that Vg-2 gene expression has a pivotal role in regulating host-seeking behaviour in young Ae. albopictus females. The identification of a molecular mechanism regulating host-seeking behaviour in mosquitoes could pave the way for novel vector control strategies aiming to reduce the biting activity of mosquitoes. From an evolutionary perspective, this is the first demonstration of vitellogenin genes controlling feeding-related behaviours in nonsocial insects, while vitellogenins are known to regulate caste-specific foraging and brood-care behaviours in eusocial insects. Hence, this work confirms the key role of vitellogenin in controlling feeding-related behaviours in distantly related insect orders, suggesting that this function could be more ubiquitous than previously thought.

Experimental Vertical Transmission of Chikungunya Virus by Brazilian and Florida Aedes Albopictus Populations.

Chikungunya virus (CHIKV) is a vector-borne alphavirus transmitted by the bites of mosquitoes, specifically infected, female mosquitoes of the invasive Aedes species. In nature, CHIKV can be maintained by vertical transmission, a phenomenon that relates to the transfer of CHIKV from the infected parent to their offspring within the ovary or during oviposition. In the present study, we conducted laboratory experiments to determine vertical transmission with Ae. albopictus populations from Brazil and Florida. Parental Ae. albopictus females were orally infected with the emergent Asian genotype of CHIKV in the first gonotrophic cycle (infectious blood meal) and tested for vertical transmission following the second (non-infectious blood meal) gonotrophic cycle. CHIKV infection and CHIKV viral titer in parental females were significantly related to population origin, with Brazilian Ae. albopictus showing higher viral dissemination and viral titer than the Florida population. Experimental vertical transmission of CHIKV was documented in one pool of female and four pools of male Ae. albopictus from Brazil (minimum infection rate, MIR, of 0.76% and 2.86%, respectively, for females and males). For the Florida population of Ae. albopictus, only one pool of males was positive for CHIKV infection, with an MIR of 1.06%. Our results demonstrate that Ae. albopictus populations from Brazil and Florida show heterogeneous CHIKV dissemination and vertical transmission, which may contribute to the epidemiology of CHIKV and may be particularly relevant to virus survival during inter-epidemic periods.

Beauveria bassiana infection reduces the vectorial capacity of Aedes albopictus for the Zika virus

Zika virus (ZIKV), a mosquito-borne flavivirus, poses a serious threat to public health worldwide, and Aedes albopictus is one of its vectors. To evaluate the potential of the entomopathogenic fungus Beauveria bassiana for ZIKV vector control, we compared the vectorial capacity of Ae. albopictus females blood-fed with ZIKV with or without exposure to Beauveria bassiana. We found that fungal infection significantly decreased the amount of ZIKV by 3.6-, 12.3- and 7.8-fold in mosquito midguts, heads and salivary glands, respectively. Similarly, fungal infection also reduced the rates of ZIKV dissemination, potential transmission and potential population transmission for mosquitoes by 26.8%, 38.4% and 35.2%, respectively. On the other hand, the median survival time and fecundity of fungus-infected mosquitoes were reduced by 84.2% and 39.8% in comparison with those of the non-fungus-infected mosquitoes. The first gonotrophic cycle length was increased by 15.3% because of fungal infection. This study revealed that B. bassiana infection significantly reduced the vectorial capacity of A. albopictus for ZIKV, which suggested that B. bassiana could be broadly and efficiently used in the field for the control of Zika vectors.

Spermathecal Filling in Aedes aegypti and Aedes albopictus: Effects of Female and Male Body Sizes and Species.

The mosquitoes Aedes aegypti (L.) (Diptera: Culicidae) and Aedes albopictus (Skuse) (Diptera: Culicidae) are ecologically similar species that have evolved independently in their native ranges, Ae. aegypti in Africa and Ae. albopictus in Asia. Where their invasive ranges overlap, interspecific mating is facilitated by both species swarming to mate around bloodmeal hosts during daylight. Here, we test hypotheses to account for variation in spermathecal filling in females of Ae. aegypti and Ae. albopictus, particularly, the effect of mosquito size on the number of spermathecae containing sperm after intraspecific and interspecifc matings of these two species. Results show that significantly more spermathecae contained sperm in large than in small Ae. albopictus females, but there was no effect of Ae. aegypti female body size on the average number of spermathecae filled. Among inseminated females, there was no effect of male size or cross-type on number of spermathecae with sperm. Differences in the effect of female size on the number of spermathecae that store sperm in Ae. aegypti and Ae. albopictus imply differences in the mating biology of these two species.

PiRNA Profiling of Dengue Virus Type 2-Infected Asian Tiger Mosquito and Midgut Tissues.

The Asian tiger mosquito, Aedes albopictus, is a competent vector for the majority of arboviruses. The mosquito innate immune response is a primary determinant for arthropod-borne virus transmission, and the midgut is the first barrier to pathogen transmission. Mosquito antiviral immunity is primarily mediated by the small interfering RNA pathway. However, the roles that the P-element induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway play in antiviral immunity in Ae. albopictus and its midgut still need further exploration. This study aimed to explore the profiles of both viral-derived and host-originated piRNAs in the whole body and midgut infected with Dengue virus 2 (DENV-2) in Ae. albopictus, and to elucidate gene expression profile differences of the PIWI protein family between adult females and their midguts. A deep sequencing-based method was used to identify and analyze small non-coding RNAs, especially the piRNA profiles in DENV-2-infected Ae. albopictus and its midgut. The top-ranked, differentially-expressed piRNAs were further validated using Stem-loop qRT-PCR. Bioinformatics analyses and reverse-transcription PCR (RT-PCR) methods were used to detect PIWI protein family members, and their expression profiles. DENV-2 derived piRNAs (vpiRNA, 24–30 nts) were observed in both infected Ae. albopictus and its midgut; however, only vpiRNA in the whole-body library had a weak preference for adenine at position 10 (10A) in the sense molecules as a feature of secondary piRNA. These vpiRNAs were not equally distributed, instead they were derived from a few specific regions of the genome, especially several hot spots, and displayed an obvious positive strand bias. We refer to the differentially expressed host piRNAs after DENV infection as virus-induced host endogenous piRNAs (vepiRNAs). However, we found that vepiRNAs were abundant in mosquito whole-body tissue, but deficient in the midgut. A total of eleven PIWI family genes were identified in Ae. albopictus; however, only AalPiwi5–7 and AalAgo3(1–2) were readily detected in the midgut. The characteristics of piRNAs in DENV-2-infected Ae. albopictus adult females were similar to those previously described for flavivirus infections but were not observed in the midgut. The reduced levels of vepiRNAs and incomplete expression of PIWI pathway genes in midgut samples from DENV-2-infected Ae. albopictus suggests that viral regulation of host piRNAs may not be an important factor in the midgut.