Enhanced esterase activity in salivary gland and midgut of Aedes aegypti mosquito infected with dengue-2 virus

Mosquitoes were infected by intrathoracic inoculation. About 95% head squashes were positive for dengue virus antigen on the 15th post infection day (PID). Esterase activity was determined in the homogenates prepared from the salivary glands and midguts on different PIDs of dengue virus inoculated and control mosquitoes showed that it was consistently higher in the virus-infected batches.     

Vertical transmission of hepatitis B virus in Culex quinquefasciatus

An investigation of the vertical transmission of hepatitis B virus (HBV) in Culex quinquefasciatus Say revealed the presence of low levels of the virus in adult F1 progeny from the first ovarian cycle of mosquitoes infected by feeding on HBV positive human blood. HBV was not transmitted vertically during the second, third and fourth ovarian cycles nor to the F2 generation. The salivary glands, ovaries and faeces of the F1 generation did not contain detectable levels of HBV. Progeny of female Cx quinquefasciatus mated with F1 males were negative for HBV.     

Epidemiological studies on Japanese encephalitis in Kyoto City area, Japan. II. Annual patterns of virus dissemination on virus recoveries from unfed Culex tritaeniorhynchus summorosus.

Annual patterns of dissemination of Japanese encephalitis virus in vector mosquitoes have been investigated at the main collection station from 1965 through 1973 at some other stations from 1969 through 1971. The virus was recovered usually from Culex tritaeniorhynchus summorosus during a period of about a month from July to August every year till 1969, and from August to September after 1970, although at some of the stations the virus was recovered intermittently for longer or shorter terms. Higher infection rates were recorded with the mosquitoes caught at the stations near to pig sheds than at the stations far from pig sheds. The infection rates at the peak of virus recovery in high epidemic year (1965 to 1967) were higher, being over 2%, than those in lower or latent epidemic years (1968 to 1973). Human patients of Japanese encephalitis were found in 17 to 20 days after the appearance of the highest peak of the infection rate in mosquitoes.     

Plasmodium gallinaceum: antibodies to circumsporozoite protein prevent sporozoites from invading the salivary glands of Aedes aegypti.

A circumsporozoite protein-specific monoclonal antibody (N2H6D5) was injected into malaria-infected mosquitoes to determine its effect on the sporogonic cycle. After injection of antibody into mosquitoes (100 ng each), positive immunofluorescence (measured on air-dried sporozoites) reactions in hemolymph extracts were observed at a dilution of 1:1000. At 72 hr postinjection the levels dropped to 1:10. Sporozoites coinjected with antibody did not invade the salivary glands. In naturally infected mosquitoes, sporozoites were released over a period of 3 to 4 days. Therefore, mosquitoes were injected twice. The first injection was a day before the beginning of sporozoite release and the second, 2 days later. Sporozoite invasion of the salivary glands was assessed 3 days after the second injection, by microscopic examination of dissected glands. At this stage, all oocysts had completed maturation and released the sporozoites. Salivary gland infections were totally prevented in mosquitoes given two injections of 100 ng N2H6D5. Hence, sustained presence of anti-circumsporozoite antibodies in the hemolymph can render female Aedes aegypti refractory to Plasmodium gallinaceum.     

Dengue 3 virus distribution in the mosquito Aedes aegyptl: an immunocytochemical study

Abstract. The dissemination of dengue (DEN) 3 virus in parenterally infected female Aedes aegypti mosquitoes was studied imrnunocytochemically. Antigen was first detected in fat body cells near the thoracic site of virus inoculation. The intussuscepted foregut, salivary glands and nervous tissue were the first major tissues infected. Nervous tissue appeared to be the primary site of amplification. Muscles, tracheae, Malphigian tubules and the posterior midgut did not become infected. The only part of the reproductive system to be infected was the calyx (71% of specimens 16–22 days post-infection) consistent with low rates of vertical transmission. After 7 days post-inoculation the salivary glands of 100% of the specimens examined were infected. Virus dissemination was slow and the most common sequence of infection following intrathoracic inoculation was as follows: thoracic fat body, intussuscepted foregut, salivary glands, cardial epithelium, thoracic ganglion, brain, compound eye, anterior midgut, intermediate midgut/anterior abdominal ganglia, and calyx/hindgut/posterior abdominal ganglia. Fat body and intussuscepted foregut tissues lost infections after 16 days post-inoculation.     

Starvation at the larval stage increases the vector competence of Aedes aegypti females for Zika virus

Aedes aegypti is the primary vector of Zika virus (ZIKV), a flavivirus which typically presents itself as febrile-like symptoms in humans but can also cause neurological and pregnancy complications. The transmission cycle of mosquito-borne arboviruses such as ZIKV requires that various key tissues in the female mosquito get productively infected with the virus before the mosquito can transmit the virus to another vertebrate host. Following ingestion of a viremic blood-meal from a vertebrate, ZIKV initially infects the midgut epithelium before exiting the midgut after blood-meal digestion to disseminate to secondary tissues including the salivary glands. Here we investigated whether smaller Ae. aegypti females resulting from food deprivation as larvae exhibited an altered vector competence for blood-meal acquired ZIKV relative to larger mosquitoes. Midguts from small ‘Starve’ and large ‘Control’ Ae. aegypti were dissected to visualize by transmission electron microscopy (TEM) the midgut basal lamina (BL) as physical evidence for the midgut escape barrier showing Starve mosquitoes with a significantly thinner midgut BL than Control mosquitoes at two timepoints. ZIKV replication was inhibited in Starve mosquitoes following intrathoracic injection of virus, however, Starve mosquitoes exhibited a significantly higher midgut escape and population dissemination rate at 9 days post-infection (dpi) via blood-meal, with more virus present in saliva and head tissue than Control by 10 dpi and 14 dpi, respectively. These results indicate that Ae. aegypti developing under stressful conditions potentially exhibit higher midgut infection and dissemination rates for ZIKV as adults, Thus, variation in food intake as larvae is potentially a source for variable vector competence levels of the emerged adults for the virus.     

Infection of strawberry flowers by Botrytis cinerea and its relevance to grey mould development

Corn stunt spiroplasma (CSS) multiplied in all injected Dulbulus maidis, reaching titres of over 1 × 10⁶ colony forming units (cfu)/insect and 1 × 10⁴ cfu/salivary gland of each insect. Spiroplasmas could be isolated from the haemolymph and from the salivary glands 1 h after injection and at any time subsequently. Insect extract at a concentration greater than the equivalent of 0.1 insects/ml was inhibitory to the growth of CSS in cultures. Helices could be seen in the haemolymph at any time after injection. However, distorted or partially deformed cells and small aggregates were not present until 2–3 wk after injection. The salivary gland cells of injected insects contained membrane-bound ‘pockets’ or ‘colonies’ packed with pleomorphic organisms, which included some filamentous forms. Intracellular colonies were always on the periphery of cells and were easily detectable by fluorescent microscopy. Both pleomorphic and filamentous forms were also seen intercellularly in the salivary glands. Following injection, transmission of CSS to maize and to sterile feeding solution were compared using 1 day feeding periods. A proportion of injected leafhoppers began to transmit to maize by the third day following injection (5%) and reached a maximum of 72% by day 14. By day 9 , 82% of the population had transmitted at least once to plants and by day 12 , 100% had transmitted. Similar insects transmitted through membranes to sterile feeding solution on day 4 (3%) reaching a maximum of 62% by day 14.     

斯氏按蚊生长发育中唾液腺配子体激活因子的变化

为探讨斯氏按蚊生长发育过程中唾液腺抽提物配子体激活因子的消长,应用体外雄配子体出丝观察方法比较羽化后未吸血及吸血组雌性斯氏按蚊唾液腺抽提物中配子体激活因子对柏氏疟原虫雄配子体出丝诱导活性的动态变化。羽化后未吸血组按蚊唾液腺配子体激活因子活性与按蚊生长、发育呈同步变化。羽化后当日至羽化后第6 d,吸血组按蚊唾液腺抽提物的GAF活性变化与未吸血组相似,吸血后该组GAF活性下降,羽化后14 d恢复到吸血前水平。吸血后斯氏按蚊唾液腺配子体激活因子活性的降低与蚊卵发育可能相关。     

Oral Susceptibility of Singapore Aedes (Stegomyia) aegypti (Linnaeus) to Zika Virus

Background

Zika virus (ZIKV) is a little known flavivirus that caused a major outbreak in 2007, in the South-western Pacific Island of Yap. It causes dengue-like syndromes but with milder symptoms. In Africa, where it was first isolated, ZIKV is mainly transmitted by sylvatic Aedes mosquitoes. The virus has also been isolated from Ae. aegypti and it is considered to be the vector involved in the urban transmission of the virus. Transmission of the virus by an African strain of Ae. aegypti has also been demonstrated under laboratory conditions. The aim of the present study is to describe the oral susceptibility of a Singapore strain of Ae. aegypti to ZIKV, under conditions that simulate local climate.

Methodology/Principal Findings

To assess the receptivity of Singapore''s Ae. aegypti to the virus, we orally exposed a local mosquito strain to a Ugandan strain of ZIKV. Upon exposure, fully engorged mosquitoes were maintained in an environmental chamber set at 29°C and 70–75% RH. Eight mosquitoes were then sampled daily from day 1 to day 7, and subsequently on days 10 and 14 post exposure (pe). The virus titer of the midgut and salivary glands of each mosquito were determined using a tissue culture infectious dose₅₀ (TCID₅₀) assay. High midgut infection and salivary gland dissemination rates were observed. By day 5 after the infectious blood meal, ZIKV was found in the salivary glands of more than half of the mosquitoes tested (62%); and by day 10, all mosquitoes were potentially infective.

Conclusions/Significance

This study showed that Singapore''s urban Ae. aegypti are susceptible and are potentially capable of transmitting ZIKV. The virus could be established in Singapore should it be introduced. Nevertheless, Singapore''s current dengue control strategy is applicable to control ZIKV.     

Salivary gland proteins of the mosquito Culex quinquefasciatus

Several properties of the salivary glands of Culex quinquefasciatus mosquitoes were analysed. The amount of protein in female salivary glands increased from 0.26 microg on day one after emergence to about 1.4 microg on day seven. The major polypeptides found in the female salivary glands had molecular weights of 35.7, 28.3, and 20.5 kDa. Antibodies produced by mice immunized by bites of Culex quinquefasciatus female mosquitoes reacted with the 35.7 and 28.3 kDa polypeptides, showing that these molecules were secreted by mosquitoes during blood feeding. The salivary glands of C. Quinquefasciatus females displayed the same morphological and biochemical organization as that of Aedes aegypti mosquitoes, accumulating apyrase in the distal portions and alpha-glucosidase in the proximal portions of the gland. Arch.     

Oral susceptibility of Aedes albopictus to dengue type 2 virus: a study of infection kinetics, using the polymerase chain reaction for viral detection

Female Aedes albopictus mosquitoes, aged 1 week, were infected with DEN-2 dengue virus. The kinetics of infection in mosquito brain and mesenteron were monitored using DNA probes with polymerase chain reaction (PCR) amplification of target DNA sequences coding for DEN-2 virus envelope protein, compared with the standard immunofluorescence assay technique (IFA). Rates of virus detection in the mesenteron of orally infected mosquitoes rose to 38% by day 4 post-inoculation, then declined until day 8, followed by irregular peaks around days 11-14 and subsequently. In mosquito head squashes, virus was detected from day 4 onwards, reaching 38% positive by day 18. Salivary glands of all the same females were found to be positive for virus by day 8 onwards. Parenterally infected Ae.albopictus females were all positive for DEN-2 in the brain and salivary glands 8 days post-inoculation. In every case, results obtained with the PCR matched those from the IFA. Our DNA probe with PCR procedure can therefore be utilized as a sensitive and reliable method for studies of DEN-2 vectors.     

Corn Stunt Spiroplasma in the Leafhopper Euscelidius variegatus

Corn stunt spiroplasma (CSS) multiplied in all leafhoppers Euscelidius variegatus injected with a culture of CSS, reaching titres of over 1x10⁶ colony forming units (cfu) per insect and 2x10⁴ cfu per salivary gland of each insect. CSS could be isolated from the haemolymph and the salivary glands at any time after injection. The growth of CSS in culture was inhibited by insect extract at concentrations greater than the equivalent of 0.1 insect/ml. Transmission of CSS to sterile feeding solution and to broad beans were compared using 24 h feeding periods. A porportion of 1.7 % of injected leafhoppers began to transmit to sterile feeding solution through membranes by the 4th day after injection, and reached a maximum of 30 % by day 14. Similar insects started transmitting to broad bean plants on day 12 (2 %), reaching a maximum of 7.5 % by day 14. The number of spiroplasmas transmitted by each insect to sterile feeding solution increased from 3, cfu on day 4 to a maximum of 80 cfu by day 14. Helices were seen in the haemolymph at any time after injection. However, partially deformed cells were not present until the 1st week and clumps of 3–4 cells and small aggregates until the 3rd week after injection. The salivary glands of injected insects contained membrane-bound “pockets” or “colonies” packed with pleomorphic, filamentous (helical and non-helical) cells and aggregates. Intracellular colonies were always at the periphery of the acini and were easily detectable by fluorescence microscopy after staining with a DNA-binding fluorescent stain. Pleomorphic and filamentous cells were also seen intercellularly in the salivary glands.     

Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells

Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, Anopheles stephensi. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control.     

Development and ageing of the salivary glands of adult male Aedes aegypti (L.) and Aedes togoi (theobald) mosquitoes (Dipteria : Culcidae)

The salivary glands of adult male Aedes aegypti and Aedes togoi (Diptera : Culicidae), varying in age from less than 1 day after emergence to 42 days or 33 days respectively, were examined by light microscopy. Following emergence, the trilobed glands rapidly accumulate secretory product and attain their full size within about 48 hr. The amount of secretion in fully developed glands shows marked individual variation, but it is predominantly located in the posterior parts of the glands. Ageing changes begin to appear after about 8 days (A. aegypti) or 10–11 days (A. togoi), and although there is wide individual variation in their extent, in general, they become progressively more severe until at the age of 4–5 weeks all mosquitoes are substantially affected. Since other tissues, including ganglia, also regress with age, it is unlikely that salivary gland changes are the prime cause of death.     

Infection of Saimiri boliviensis monkeys with Plasmodium coatneyi

Abundant, apparently normally developing, liver-stage parasites of Plasmodium coatneyi were demonstrated following injection of sporozoites dissected from the salivary glands of Anopheles dirus mosquitoes. Erythrocytic development was not demonstrated.     

The effects of blood feeding and exogenous supply of tryptophan on the quantities of xanthurenic acid in the salivary glands of Anopheles stephensi (Diptera: Culicidae)

Xanthurenic acid (XA), produced as a byproduct during the biosynthesis of insect eye pigment (ommochromes), is a strong inducer of Plasmodium gametogenesis at very low concentrations. In previous studies, it was shown that XA is present in Anopheles stephensi (Diptera: Culicidae) mosquito salivary glands and that during blood feeding the mosquitoes ingested their own saliva into the midgut. Considering these two facts together, it is therefore likely that XA is discharged with saliva during blood feeding and is swallowed into the midgut where it exerts its effect on Plasmodium gametocytes. However, the quantities of XA in the salivary glands and midgut are unknown. In this study, we used high performance liquid chromatography with electrochemical detection to detect and quantify XA in the salivary glands and midgut. Based on the results of this study, we found 0.28+/-0.05 ng of XA in the salivary glands of the mosquitoes, accounting for 10% of the total XA content in the mosquito whole body. The amounts of XA in the salivary glands reduced to 0.13+/-0.06 ng after mosquitoes ingested a blood meal. Approximately 0.05+/-0.01 ng of XA was detected in the midgut of nonblood fed An. stephensi mosquitoes. By adding synthetic tryptophan as a source of XA into larval rearing water (2 mM) or in sugar meals (10 mM), we evaluated whether XA levels in the mosquito (salivary glands, midgut, and whole body) were boosted and the subsequent effect on infectivity of Plasmodium berghei in the treated mosquito groups. A female specific increase in XA content was observed in the whole body and in the midgut of mosquito groups where tryptophan was added either in the larval water or sugar meals. However, XA in the salivary glands was not affected by tryptophan addition to larval water, and surprisingly it reduced when tryptophan was added to sugar meals. The P. berghei oocyst loads in the mosquito midguts were lower in mosquitoes fed tryptophan treated sugar meals than in mosquitoes reared on tryptophan treated larval water. Our results suggest that mosquito nutrition may have a significant impact on whole body and midgut XA levels in mosquitoes. We discuss the observed parasite infectivity results in relation to XA's relationship with malaria parasite development in mosquitoes.     

Alterations in polypeptides pattern in malaria vector Anopheles stephensi, fed upon immunized blood causing fecundity reduction

Changes in polypeptides pattern of haemolymph, midgut, ovary and salivary glands of female mosquito A. stephensi were studied when fed upon anti-mosquito haemolymph antibodies. The expression of almost all polypeptides was reduced in haemolymph and ovary of the immune fed mosquitoes as compared to control. However, there was no significant difference in case of midgut and salivary glands. Seven polypeptides 100, 90, 84, 80, 62, 19 and 12.5 kDa were absent in haemolymph and five 92, 90, 80, 60 and 55 kDa were absent in ovaries. Changes in the polypeptide pattern have been correlated with the fecundity reduction due to immunized blood feeding.     

Transovarial Transmission of Chikungunya Virus by Aedes albopictus Mosquitoes Ingesting Microfilariae of Dirofilaria immitis under Laboratory Conditions

Female Aedes albopictus mosquitoes of the Miki strain were experimentally fed on defibrinated sheep blood containing 5× 10⁷ PFU of chikungunya virus and 20,000 microfilariae of Dirofilaria immitis per milliliter. Fully engorged mosquitoes transmitted the virus to a small percentage of the F1 progeny, but females of the F1 generation did not transmit the virus to the F2 progeny. The control mosquitoes that ingested the virus without microfilariae did not transmit the virus to their eggs, larvae, or pupae in the F1 or F2 generations. These results showed that A. albopictus of this strain that concurrently ingested the virus and microfilariae transmitted the virus by the transovarial route under experimental conditions.     

Studies on two strains of Plasmodium cynomolgi in New World and Old World monkeys and mosquitoes

Infections that cause the Gombak and Smithsonian strains of Plasmodium cynomolgi were induced in Macaca mulatta, Aotus lemurinus griseimembra, Aotus nancymai, and Saimiri boliviensis monkeys. Transmission of the Gombak strain to Aotus spp. monkeys was obtained by the injection of sporozoites dissected from the salivary glands of experimentally infected Anopheles dirus and by the bites of infected An. dirus and Anopheles farauti mosquitoes. Two S. boliviensis monkeys were infected via the injection of sporozoites dissected from An. dirus. Prepatent periods in New World monkeys ranged from 14 to 44 days, with a median of 18 days. The Smithsonian strain was transmitted via sporozoites to 1 A. lemurinus griseimembra and 9 A. nancymai monkeys. Prepatent periods ranged from 12 to 31 days.     

Fluid transport across the ducts of the salivary glands of a mosquito

We compared the volumes of fluid expelled from the salivary stylets of mosquitoes having ducts transected at different levels. An oil-filled apparatus was devised to measure salivary output. About $\text{1}\text{10}$ of normal salivary volume was produced by mosquitoes when ducts were transected and externalized just anterior to the salivary glands. Such mosquitoes ejected more saliva than could be contained within the walls of the ducts. Transection posterior to the pump prevented salivation entirely. Skin reactive antigen was not detected (as a wheal) when mosquitoes with transected ducts fed on man. We conclude that fluid is transported across the walls of the ducts that drain the salivary glands of a mosquito.