Factors influencing the abundance of Japanese encephalitis vectors in ricefields in India – I. Abiotic

Mosquitoes of the Culex vishnui subgroup (Diptera: Culicidae) are the most important vectors of Japanese encephalitis (JE) and ricefields are their most productive breeding sites in south India, where predominant species of this subgroup are Culex tritaeniorhynchus Giles, Cx. pseudovishnui Colless and Cx. vishnui Theobald sensu stricto. The relationship of 13 abiotic variables with the abundance of Cx. vishnui subgroup immatures was investigated in transplanted rice fields for 3 years (1991-94) covering three different crop seasons. The results from the multiple regression model suggested paddy height (- ve), water temperature (+ ve), dissolved oxygen (- ve), ammonia nitrogen (- ve) and nitrate nitrogen (+ ve) to be the best predictor variables associated with the immature abundance, nearly always consistent in their effects within and between seasons. Application of synthetic nitrogenous fertilizers to the rice fields was followed by a rise in concentration of ammonia nitrogen and a subsequent increase in nitrate nitrogen level in the rice field water, during which an increase in the density of larval instars was observed.     

Host preference of mosquito vectors of Japanese encephalitis.

The host preference of 4 Culex mosquito species collected in Miaoli and Pingtung counties, Taiwan was studied by capillary precipitin method. Antisera to alum-precipitated sera of man, bovine, swine, rabbit, horse, dog, cat, mouse, chicken, duck, and pigeon were produced in rabbits and reacted with 758 mosquito blood meals among which reactions to one or more antisera. Culex annulus and Culex tritaeniorhynchus summorosus showed a great avidity for pig, and Culex fuscocephala for bovine. Culex pipiens fatigans was ornithophilic. None of 110 C. t. summorosus and 2.4% of 223 C. annulus had fed on man. Among 66 samples of C.p. fatigans tested 10.3% had fed on man, while none of 359 C. fuscocephala did. It seems that the latter does not act as a primary vector of Japanese encephalitis.     

Thermostability of Japanese encephalitis vaccine produced in India.

Different batches of bulk vaccine, final bulk at in-process level, finished freeze-dried and reconstituted Japanese encephalitis vaccine were assayed for their stability at temperatures of 22, 37 and 40 degrees C. After ultrazonal purification of 50 times concentrated brain suspension, JE Bulk vaccine was found to be stable for up to 2 years at 4 degrees C, however, the percentage loss in potency (log 10 N antibody titre) after 2.5 years was 24%. Three-times concentrated final bulk showed rapid deterioration by the fourth week at 37 and 40 degrees C. Freeze-dried JE vaccine maintained at 22 degrees C for 28 weeks did not show perceptible deterioration. At 37 degrees C, the same vaccine started showing deterioration (14%) after 8 weeks whereas at 40 degrees C the loss of potency was 24% after 8 weeks. The freeze-dried vaccine was found to be stable for up to 2 weeks duration at 40 degrees C.     

Factors influencing the abundance of Japanese encephalitis vectors in ricefields in India--II. Biotic

The relationship of insect predators and phytoplankton with the abundance of Culex tritaeniorhynchus Giles, Cx. vishnui Theobald and Cx. pseudovishnui Colless mosquito larvae and pupae (Diptera: Culicidae) in ricefields was investigated during three rice growing seasons. Notonectids were the most abundant insect predators, whereas diatoms dominated among phytoplankton. Multiple regression analysis showed that the occurrence of notonectids (both nymphs and adults) was negatively associated with larval abundance. Phytoplankton, especially diatoms and blue green algae (BGA), were found to favour abundance of culicine immatures during Navarai and Kuruvai crops, respectively. Larval gut analysis showed that the intake of algae by late instars was high, with 93%, 58% and 24% of diatoms, BGA and green algae, respectively. Filamentous algae may not necessarily be of nutritive value, but they are observed to form mats, which provide protection to the mosquito immatures from the predators.     

Preventive strategies for frequent outbreaks of Japanese encephalitis in Northern India

Japanese encephalitis (JE) remains the most important cause of acute viral encephalitis and continues to spread to hitherto unaffected regions like Indonesia, Pakistan and Australia. Approximately 60% of the world population inhabits JE endemic areas. Despite its restricted range mostly in the developing countries, a high annual incidence of 50,000 cases and about 10,000 deaths has been reported. Disease can be fatal in 25% cases. Magnitude of the problem is even more alarming since the survivors are left with serious long-term neuropsychiatric sequelae. Almost every two years, epidemics of JE occur in Indian subcontinent with a high mortality. JE virus infection results in different disease manifestations in host from mild subclinical febrile illness to clinical infections leading to encephalitis. No antiviral treatment is so far available for JE. The prevention of JE can be achieved by controlling the vector or by immunization regime. The vector control in the rural areas, which are the worst affected ones, is practically almost impossible. Three vaccines that have been implicated against JE include inactivated mouse brain derived, inactivated cell culture derived and cell culture derived live attenuated JE vaccine. But each has its own limitation. Currently, attempts to synthesize recombinant DNA vaccine are being made. New therapeutics are on the way of development like use of minocycline, short interfering RNA, arctigenin, rosmarinic acid, DNAzymes etc. However, the immune mechanisms that lead to JE are complex and need to be elucidated further for the development of therapeutics as well as safe and efficacious JE vaccines.     

The role of pre- and post-imaginal experience in the host-finding and oviposition behaviour of the cabbage moth

The influence of pre- and post-imaginal experience on the orientation, landing, and oviposition of the female cabbage moth, Mamestra brassicae (L.) (Lepidoptera: Noctuidae) was studied in a wind tunnel. The results of experiments on the influence of pre-imaginal experience do not support the Hopkins host selection hypothesis because the females’ responses to host plants were not affected by the plant species used for feeding the larvae. The females were initially attracted to and landed on chrysanthemum whether or not they were fed on this plant species. In addition, the oviposition preference for cabbage plants was not changed by the larval feeding regimen. Imaginal experience influenced the females’ subsequent orientation/landing responses: exposure of the females to chrysanthemum plants for 5 min decreased their subsequent responses to this species, but subsequent responses to cabbage were not changed when the females were exposed to cabbage plants. Overnight exposure of females to a chrysanthemum or cabbage plant decreased the subsequent orientation/landing on the same species offered in non-choice tests. A similar effect was observed in responses to chrysanthemum plants after overnight contact, and possibly to cabbage plants, in the two-choice tests. Overnight exposure to cabbage or chrysanthemum had no effect on subsequent oviposition: females from both treatments oviposited more often on cabbage.     

Blue-green algae from rice fields in Kerala State,India

A survey of blue-green algae occurring in rice fields in Kerala State (India), has been carried out. Among the thirty taxa identified and described, ten are recorded for the first time in a rice-field environment. The taxa included unicellular, non-heterocystous filamentous, heterocystous filamentous and heterotrichous forms.Dedicated to Prof. T. V. Desikachary on his 65th Birthday.     

Spider predation on a mirid pest in Japanese rice fields

Spiders are common generalist predators, and understanding their potential in biological control is important for the development of integrated pest management programs. In this study, predation by three groups of spiders on the mirid bug Stenotus rubrovittatus (Hemiptera: Miridae) in rice paddies was investigated using DNA-based gut-content analysis. A laboratory feeding study revealed that the detection half-lives of bug DNA in the spider gut at 25 °C was 3.4 days for Lycosidae and 1.5 days for Tetragnathidae. Individual spider predation on the mirid bug was investigated by detecting DNA of prey in field-collected spiders. In total, 1199 spiders were assayed from three spider groups: Pirata subpiraticus (Lycosidae), Tetragnatha spp. (Tetra-gnathidae), and Pachygnatha clercki (Tetra-gnathidae), which each differ in their preferred microhabitat as well as their predatory habits. Detection rates of prey DNA in spiders increased significantly with the density of prey across all spider groups. P. subpiraticus and Tetragnatha spp. predation showed a better fit to a saturated response curve to increasing prey density, while P. clercki showed a simple linear relationship with prey density. Densities of alternative prey species did not affect the detection rates of mirids. These results suggest that predation on pests by generalist predators in an agroecosystem is affected not only by prey abundance but also by predator preference for specific prey. Predator preference is therefore an important factor to consider when estimating the role of natural enemies as biological control agents.     

Ammonia volatilization losses from prilled urea,urea supergranules (USG) and coated USG in rice fields

Summary About 8.4 per cent of applied nitrogen was lost as ammonia during a week after application when prilled urea was broadcast or banded and incorporated in soil 20 days after sowing of rice. Ammonia volatilization was reduced to 3.3 per cent when urea supergranules (USG) were used. Coating of USG with DCD or neem cake showed no advantage. Ammonia volatilization was only 0.7 to 1.6 per cent when fertilizer was applied at panicle initiation stage of rice; highest values were again obtained with prilled urea. The experiments were carried out in closed cages.     

Generation and immunogenicity of Japanese encephalitis virus envelope protein expressed in transgenic rice

Transgenic plants have become attractive as bioreactors to produce heterologous proteins that can be developed as edible vaccines. In the present study, transgenic rice expressing the envelope protein (E) of Japanese encephalitis virus (JEV), under the control of a dual cauliflower mosaic virus (CaMV 35S) promoter, was generated by Agrobacterium-mediated transformation. Southern blot, Northern blot, Western blot and ELISA analyses confirmed that the E gene was integrated into transgenic rice and was expressed in the leaves at levels of 1.1-1.9 μg/mg of total soluble protein. After intraperitoneal immunization of mice with crude protein extracts from transgenic rice plants, JEV-specific neutralizing antibody could be detected. Moreover, E-specific mucosal immune responses could be detected in mice after oral immunization with protein extracts from transgenic rice plants. These results show the potential of using a transgenic rice-based expression system as an alternative bioreactor for JEV subunit vaccine.     

Re-evaluation of the role of corpora cardiaca in calling and oviposition behaviour of giant silk moths

Re-investigation of the role of the corpora cardiaca in the reproductive behaviour of the giant silkmoths, Hyalophora cecropia and Antheraea polyphemus, showed that this pair of glands plays no essential role, either in “calling” behaviour by virgin females or in increased oviposition due to mating. Removal of corpora cardiaca-corpora allata complexes, either from diapausing pupae or from freshly eclosed adult females, had no effect on the calling behaviour or on its timing in either species. Moreover after mating, these operated females laid eggs in the typical mated oviposition pattern. Furthermore, females in which there was only a nervous connection between the brain and the abdomen but no haemolymph circulation called normally and oviposited after mating.Although the corpora cardiaca were not essential for calling behaviour, hormogenates of corpora cardiaca-corpora allata complexes and blood from calling or ovipositing females induced a typical “calling” response in 30–60% of the isolated virgin H. cecropia abdomens tested. This activity was not species-specific as it was also found in Manduca sexta, but the restriction of major activity to corpora cardiaca extracts and haemolymph suggested that a neurosecretory factor may modulate the normal neural control of calling behaviour.     

Quantitative genetics of oviposition behaviour and interactions among oviposition traits in the sand cricket

We performed a quantitative genetic study of oviposition behaviours and oviposition traits in the sand cricket Gryllus firmus. Egg survival in crickets depends on the depth at which they are inserted into the soil with the ovipositor. We examined whether egg depth depends on ovipositor length alone, or on both morphological and behavioural traits associated with oviposition. Heritability estimates were high (h² >0.5) for ovipositor length and small (h²=0.2) for oviposition behaviours. Negative genetic correlations between ovipositor length and some behavioural traits (digging depth and the behavioural component of egg depth) indicated compensation between oviposition traits on egg depth. Because of behavioural compensation, females with different ovipositor lengths subject to stabilizing selection on egg depth could have equal fitnesses. Females laid their eggs deeper, and their eggs were marginally more evenly distributed in dry than in wet sand. This suggests adaptive phenotypic plasticity in laying behaviour, but may also result from physical constraints of the substrate on the insertion of the ovipositor. The absence of significant between-family variation in oviposition traits in response to sand moisture indicates low evolutionary potential for phenotypic plasticity in oviposition traits according to soil moisture. In highly unpredictable environments, females could spread the risk of desiccation by laying eggs at different depths independently of environmental conditions (bet hedging). Our results show significant additive genetic influences on the ability of a female to spread risks as measured by genetic variation in egg distribution, suggesting that a bet-hedging strategy of egg laying has the potential to evolve in this population. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Antiviral and neuroprotective role of octaguanidinium dendrimer-conjugated morpholino oligomers in Japanese encephalitis

Background

Japanese encephalitis (JE), caused by a mosquito-borne flavivirus, is endemic to the entire south-east Asian and adjoining regions. Currently no therapeutic interventions are available for JE, thereby making it one of the most dreaded encephalitides in the world. An effective way to counter the virus would be to inhibit viral replication by using anti-sense molecules directed against the viral genome. Octaguanidinium dendrimer-conjugated Morpholino (or Vivo-Morpholino) are uncharged anti-sense oligomers that can enter cells of living organisms by endocytosis and subsequently escape from endosomes into the cytosol/nuclear compartment of cells. We hypothesize that Vivo-Morpholinos generated against specific regions of 3′ or 5′ untranslated regions of JEV genome, when administered in an experimental model of JE, will have significant antiviral and neuroprotective effect.

Methodology/Principal Findings

Mice were infected with JEV (GP78 strain) followed by intraperitoneal administration of Morpholinos (5 mg/kg body weight) daily for up to five treatments. Survivability of the animals was monitored for 15 days (or until death) following which they were sacrificed and their brains were processed either for immunohistochemical staining or protein extraction. Plaque assay and immunoblot analysis performed from brain homogenates showed reduced viral load and viral protein expression, resulting in greater survival of infected animals. Neuroprotective effect was observed by thionin staining of brain sections. Cytokine bead array showed reduction in the levels of proinflammatory cytokines in brain following Morpholino treatment, which were elevated after infection. This corresponded to reduced microglial activation in brain. Oxidative stress was reduced and certain stress-related signaling molecules were found to be positively modulated following Morpholino treatment. In vitro studies also showed that there was decrease in infective viral particle production following Morpholino treatment.

Conclusions/Significance

Administration of Vivo-Morpholino effectively resulted in increased survival of animals and neuroprotection in a murine model of JE. Hence, these oligomers represent a potential antiviral agent that merits further evaluation.     

The role of chemotactile stimuli in the oviposition preferences of Colias butterflies

Summary The legume foodplants of Colias butterflies possess specific chemical components which stimulate oviposition in females of the genus. Different legumes provide different degrees of stimulation, as demonstrated by close correlation between field observations and laboratory experiments with a new behavioral assay. Several generalizations emerge from this study. 1) Chemical preferences for various foodplants are under genetic control in C. meadii, and are at least partly independent of an individual's previous exposure to different legumes. 2) In some cases, chemical cues alone do not allow females to discriminate between species. Long-range search cues may be used to enhance discrimination in such instances. 3) Lupinus, a legume which is not usually utilized by Colias, stimulates oviposition in the laboratory, indicating its chemical affinity with other Leguminosae. The behavioral assay described should prove useful for the future identification of specific oviposition stimulants and deterrents. It is stressed, however, that chemotactile cues are involved only in the final step of oviposition, and that understanding foodplant choice in nature will require in-depth investigation into the mechanics of individual search processes.     

Different oviposition behaviour in Chrysomelid beetles: Characterisation of the interface between oviposition secretion and the plant surface

The behavioural sequence of the oviposition process can be quite distinct in herbivorous leaf beetles: eggs are either connected to the plant surface or laid into mesophyll tissue. Females of two beetle species were recorded with a digital camera in order to analyse their oviposition behaviour. The morphology of the plant-egg-interface was investigated in three species by histological studies and scanning electron microscopy. The lily leaf beetle, Lilioceris lilii, oviposits directly on the plant cuticle without damaging the plant tissue. Several eggs are released together with secretion and are laid in a row. The secretion sorbes in the plant cuticle. In the close relative, Lilioceris merdigera, the secretion can enter the leaf tissue via the stomata. The mustard leaf beetle, Phaedon cochleariae, gnaws a characteristic hole into the epidermis and part of the mesophyll. Into this cavity, it releases in an alternating sequence secretion, a single egg and again secretion. The egg, which is usually laid on plants growing in wetlands, is surrounded by an extrachorion that might possess a respiratory function. The potential eco-physiological relevance of the specific nature of the placement of eggs and secretion on or in the plant tissue is discussed.     

Effects of organic and inorganic fertilisers on mosquito populations in rice fields of southern India

The effects of nitrogenous (inorganic) fertilisers, organic manures and blue-green algae (BGA) biofertiliser on mosquito populations (Diptera: Culicidae) were studied in rice fields of Madurai, Tamil Nadu, south India, with particular attention to Culex vishnui Theobald, Cx. pseudovishnui Colless and Cx. tritaeniorhynchus Giles, the vectors of Japanese encephalitis (JE). The application of urea, a nitrogenous fertiliser, in rice fields significantly increased the grain yield and the population densities of mosquito larvae and pupae (anophelines as well as culicines) in a dose-related manner. Fields treated with inorganic fertilisers (N, P, K) had significantly higher population densities of mosquito immatures than fields treated with organic manures (farmyard manure and green manure). Without nitrogenous fertiliser, BGA increased paddy yield without enhancing mosquito production. Therefore, the use of BGA with less nitrogenous fertiliser is recommended, which is beneficial economically and agronomically to the farming community and also significantly reduces mosquito production in rice fields. Increased use of nitrogenous fertiliser over the past two decades may have contributed to the increased severity of Japanese encephalitis epidemics, vectors of which breed in rice fields.