Research Article

Laboratory Bioassay of Chilodonella uncinata, an Entomopathogenic Protozoan, against Mosquito Larvae  

Bina Pani Das1,3 , Kedar Deobhankar2 , Karuna N Pohekar3 , Rahul Marathe2 , Syed Akhtar Husain3 , P. Jambulingam4
1. National Centre for Disease Control, 22 Sham Nath Marg, Delhi 110054, India
2. Ross Lifescience Pvt. Ltd., Plot No 96, Sector no 10, PCNTDA, Bhosari, Pune 411026, India
3. Department of Biosciences, Jamia Millia Islamia University, New Delhi 110025, India
4. Vector Control Research Centre, Indira Nagar, Puducherry 605006, India
Author    Correspondence author
Journal of Mosquito Research, 2016, Vol. 6, No. 10   doi: 10.5376/jmr.2016.06.0010
Received: 19 Oct., 2015    Accepted: 18 Dec., 2015    Published: 27 Jan., 2016
© 2016 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Das B.P., Deobhankar K., Pohekar K.N., Marathe R., Husain S.A., and Jambulingam P., 2016, Laboratory bioassay of Chilodonella uncinata, an entomopathogenic protozoan, against mosquito larvae, Journal of Mosquito Research, 6(10): 1-10 (doi: 10.5376/jmr.2016.06.0010)

Background & objectives Use of microbial control agents provides alternative method for adequate insect management. We evaluated laboratory bioassay of Chilodonella uncinata, a natural protozoan parasite of mosquito larvae.
Methods Two formulations and four strains: North India (Monsoon and Pre-monsoon) strain, South India strain, updated strain of Chilodonella uncinata were tested against Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti larvae at four institutes.
Results & Interpretation Delayed development was noted in mosquito larvae exposed to Ch. uncinata formulation that produced better effect in all larval species. Efficacy of this biolarvicide is not dose dependant as least dose produced maximum mortality with minimum post exposure. An. stephensi larvae were most sensitive followed by Cx. quinquefasciatus and Ae. aegypti which required longer post exposure. Of the four strains, updated strain was most effective and stable; south India strain had rapid killing effect; Pre-monsoon strain was more effective than Monsoon strain as the later required 20x higher dose (cs/ml) to induce satisfactory mortality in An. stephensi larvae with higher values for LT50 (4.71) and LT90 (6.84) as against LT50 (2.5) and LT90 (3.57) of former strain. Tea bag formulation is easy to store, transport and treat, was found to have a shelf life of >18 months resulting in satisfactory efficacy against An. stephensi with LT50 (5.16) and LT90 (7.69) noted at 0.25 g even after 6 months of storage. These laboratory test data suggest that a lower dose of this protozoan formulation can be used as a potential biolarvide to control mosquito larvae as an alternative to chemical insecticides under integrated vector management.
Aedes aegypti; Anopheles stephensi; Chilodonella uncinata; Culex quinquefasciatus; Entomopathogenic protozoan; Mosquito larvicidal activity

1 Introduction

Mosquitoes act as vectors for most of the life threatening diseases like malaria, Japanese encephalitis, yellow fever, dengue fever, Chikungunya fever, filariasis, West Nile Virus infection, etc. Extensive use of synthetic chemical insecticides such as organochlorine and organophosphate compounds against vector mosquitoes to control malaria and other mosquito borne diseases for about four decades has not been very successful.
These diseases are not only prevalent but also outbreaks into epidemics (Mittal, 2003). Sustained and prolonged use of chemical insecticides has led to the development of insecticide resistance in the target mosquito population, severe suppression of non-target organisms and general pollution of the environment. Additional and innovative vector control methods are therefore required either alone or in combination with traditional approaches, to decrease the transmission of the disease (Rojas et al., 1987). One such method is the use of microbial control agents that are naturally occurring, comprised of microscopic living organisms or microbial pest control agents (viruses, bacteria, fungi, protozoa) or the toxins produced by these organisms (Burger, 1981). Nearly 100 species of entomophilic bacteria, over 1600 viruses, more than 750 fungi and 300 known species of entomophilic protozoa have been isolated from arthropods and more are being discovered each year.
Bacillus thuringiensis var. israelensis, is the only microbial insecticide included in Indian urban malaria control programme since 2002 and can be used in places that are usually been treated with larvicide like Temephos and Fenthion. However, B.t.i does not recycle in the environment and its effect against surface feeding Anopheline larvae is impacted by various bioenvironmental factors, thus requiring weekly application in most habitats (Mittal, 2003), increasing the end cost in the process. Most viral biopesticides developed so far encounter difficulties as they are not stable under ultra violet rays of sun and more expensive than chemical pesticides due to in vivo production (Canon, 2013).
Majority of the entomophilic protozoa are from Microsporidia and Ciliophora. Microsporidia are all intracellular parasites and there is no easy and cheap way to mass produce them in vitro. The use of insect cell cultures to mass-produce microsporidia is not yet economically feasible because of limitations on the mass culture of insect cell themselves and because the yield of microsporodia spores per infected cell is too low. Among Ciliophora, 4 species belonging to 3 genera, viz. Lambornella clarki (L. clarki), L. stegomyiae, Tetrahymena pyriformis and Chilodonella uncinata (Ch. uncinata) are known endoparasite of mosquito larvae (Das, 2003). L. clarki Corliss & Coats, a natural pathogen of the tree hole breeding mosquito, Aedes sierrensis (Ae. sierrensis) received considerable attention during last two decades of past century as a potential biological control agent for container breeding mosquitoes (Washburn, 1995; Egeter et al., 1986; Washburn and Anderson, 1986).
Chilodonella uncinata (Ehrenberg), 1838 (Subphylum: Ciliophora: Cryptophorida: Chilodonellidae), a natural biological control agent was discovered accidentally in mosquito vectors (Culex tritaeniorhynchus and Cx. pseudovishnui) of Japanese encephalitis (JE) virus breeding in an extensively paddy growing village in Haryana (Das, 2003). Chilodonella uncinata has a unique mode of behavior.  It is a facultative parasite of mosquito larvae and its distribution as a free swimming (trophont) stage is scanty. In presence of susceptible mosquito larvae, it changes itself and become parasitic in habit.  Number of them simultaneously attack one single larva at different points (head, thorax, abdomen, siphon, anal fin, antennae, etc.) and enter body cavity of the host larva by drilling through the host cuticle. Thereafter, it multiplies inside the host body at the expense of the internal viscera of the host larva and releases numerous minute motile spores, ultimately killing the larva and finally escaping the cadaver of larva (recycle in the environment) to attack fresh larvae to continue the cycle. The organism is so virulent that even a few of them can cause chronic infection leading to death in susceptible host larvae (Das, 2004). This type of mode of entry of Ch. uncinata from outside through host cuticle is similar to that reported in case of two parasitic ciliates, viz. L. stegomyiae and L. clarki and one parasitic fungi, Coelomomyces sp. (Das, 2012). Coelomomyces is known to parasitise Anopheline larvae in paddy fields in South India (Chandrahas and Rajagopalan, 1979)and in a temporary pool in North India (Gugnani et al., 1963). However, Coelomomyces has a complex life cycle and requires both the primary host (mosquito larvae) and intermediate host (copepod) for reproduction (Lacey and Lacey, 1990).
Ch. uncinata was successfully colonized under laboratory conditions of three institutes, viz. National Centre for Disease Control, earlier as National Institute of Communicable Diseases (NICD),Delhi; Department of Biosciences, Jamia Millia Islamia (JMI), New Delhi and Ross Lifescience Pvt. Ltd. (ROSS), Pune, India. Three basic strains and one formulation of Ch. uncinata were developed at NICD (2000-2007) and updated strain and its tea bag formulation were developed during 2011-2012 at JMI (Das, 2012). In the formulation the active ingredient (Ch. uncinata) remain inactivated, which when released in water: works as an effective biological control agent for mosquito vectors of human diseases (Malaria, JE and Dengue/Chikungunya); Ch. uncinata gets reconstituted after a gap of two hours to few days and search out young mosquito larvae and infect them by drilling through the larval cuticle. Initially, the active ingredient of this protozoan formulation was isolated from wild caught JE vector larvae but when formulation was prepared it was found to be more effective against larvae of malaria vector (Das, 2008). We report here the results of laboratory bioassay of Ch. uncinata against Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti larvae carried out at Vector Control Research Centre (VCRC), Puducherry; NICD, Delhi; JMI, New Delhi and ROSS, Pune, India.
2 Material and Methods
2.1 Preparation of protozoan sample
The protozoanwas cultured under laboratory conditions at NICD on artificial medium using simple technique in glass container and filtered through 10µm mesh cloth in 2000 (Das, 2004). The culture filtrate was the first strain developed and designated as Ch. uncinata NICDENTBPL 13106 (North India) monsoon strain. (ii) Ch. uncinata NICDENTBPL 13106 (North India) pre-monsoon strain was developed by isolating the protozoan from wild caught dead and diseased mosquito larvae collected from its area of influence in June 2004. (iii) Ch. uncinata NICDENTBPL 13106 (South India) strain was developed in 2005 using the protozoan parasites laid by infected Cx. tritaeniorhynchus mosquitoes brought from a mosquito colony of an institute at Madurai, Tamil Nadu, India. However, all these strains required to be sub cultured on every alternate day to avoid contamination. Later, the culture procedure was updated by addition of preservatives, etc. and (iv) an updated (Ch. uncinata BP 610) strain was developed by using the laboratory facilities at JMI and isolating these protozoan from wild caught dead and diseased Cx. tritaeniorhynchus larvae from paddy fields from Safiabad village and there was no need to sub culture this strain up-to one month under laboratory condition (Temp. 27±2℃). This strain was deposited with the International Depository Authority – ATCC, U.S.A. (Das, 2012). 
Two granular formulations, viz., Ch. uncinata NICDENTBPL 13106 North India and Ch. uncinata BP 610 were developed at NICD and JMI respectively. Granular formulations were prepared using concentration of 6.5x104 to 8.0x104 cells/ml of these culture strains with base material as sterilized sand to entrap the active ingredient i.e. the freely moving Ch. uncinata (Das, 2012). Ch. uncinata BP 610 formulation prepared at JMI was wrapped in tea bags in various dosages e.g. 0.1g, 0.25g, 0.5g, 1.0g, 2.0g, 4.0g, 8.0g, 16.0g, 32g and each one of these tea bags was sealed in a small plastic pouch for retaining moisture and easy storage (at room temperature), transport and delivery for its bio-efficacy evaluation under laboratory condition. These tea bags were also sent to ROSS by post and evaluated there 6 months later from the date of preparation of these bags at JMI. Ch. uncinata culture was established at ROSS in February 2014 from a 4.0g tea bag of Chilodonella uncinata BP 610 formulation which was prepared in Sept 2012 at JMI. This strain is being maintained at ROSS till date.
The efficacy of four strains and two granular formulations of Ch. uncinata were tested against the immatures of mosquito vectors in the laboratory at 4 institutes, viz.: i) Ch. uncinata NICDENTBPL 13106 South India strain was tested using the facilities available at VCRC in 2005; ii) formulation as well as pre-monsoon and monsoon strains of Ch. uncinata NICDENTBPL 13106 were evaluated at NICD during 2005-2007; iii) Ch. uncinata BP 610 strain and its granular formulation (in tea bags) were evaluated at JMI (2011-2012); iv) These tea bags were also sent to ROSS by post and evaluated there nearly 6 months later from the date of preparation at JMI. The strain Ch. uncinata BP 610 and various dosages of formulation (in tea bags) were obtained from JMI (2011-12) while the rest were made available from NICD during 2005-2007.
2.2 Test organisms
Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti were reared in the laboratory of Vector Control Research Centre, Pondicherry; National Centre for Disease Control and Ross Life Science Pvt Ltd. The larvae were fed in these institutes on dog biscuit and yeast powder in the 3:1 ratio, yeast tablet and yeast tablet wrapped in hand made muslin cloth bag respectively. Adults were provided with 5% glucose solution soaked in cotton pads and water soaked raisins as a source of energy and rabbit/pigeon for blood meal. Mosquitoes were kept in the insectary maintained at 28 ± 2˚C and 70-85% relative humidity.
First instar larvae of Ae. aegypti and early second instar larvae of An. stephensi and Cx. quinquefasciatus were obtained from the colonies of VCRC, NICD and ROSS. First and early second instar larvae were used as they had four to three stages before becoming adults for determining bio-larvicidal potential of test sample.
2.3 Laboratory bioassay
The bio-larvicidal activity of four culture strains of Ch. uncinata and its two formulations was evaluated as per the method recommended by WHO. While pre-monsoon and monsoon strain were used as such, South India strain was centrifuged at 10,000 rpm for 10 minutes just before initiation of the experiment at VCRC. Same protocol was followed with the updated (Ch. uncinata BP 610) strain at JMI except that it was centrifuged at 3000 rpm for 3 minutes. At the same time test larvae were also exposed to normal strains (without centrifugation) at VCRC and JMI to observe the impact of centrifugation on larval mortality. 
25 test larvae of each species were held in 500ml disposable cup containing 250 ml of chlorine free tap water. Ch. uncinata cells were counted as per numbers present per ml of culture used and tested for mortality of mosquito larvae. Concentrations ranging from 103 to 8×105 cells/ml or selected quantity of granular formulation as such or wrapped in tea bags of protozoan sample were added to the bioassay cups. For each experiment four replicates were maintained at a time. Larval food was added to the experimental cups from second day and not on the first day to avoid interference with the experiment. Bioassay cups containing larvae and larval food, but without Ch. uncinata cells/formulation served as control.  
All the test and control cups were covered with netting to prevent successfully emerged adults from escaping into the environment (WHO, 1996). Observation was made at daily interval. Larval mortality was corrected for control mortality using Abbott’s formula. Some of the dead larvae from the experimental cups were examined microscopically after a gap of 4-5 days for the protozoan (Ch. uncinata) infection. Re-isolation of the protozoan from the cadavers on artificial medium was also attempted to confirm infection.
2.4 Statistical analyses
Statistical analysis for calculating LT50 and LT90 values (in days) was done by using MS Excel software.
3 Results
Larvicidal activity of different strains and formulations of Ch. uncinata at various concentrations and dosages against first instar larvae of Ae. aegypti and early second instar larvae of An. stephensi and Cx. quinquefasciatus were expressed in terms of the percentage of larvae that were killed (% Mortality) during post exposure period (in day)s. On examination under microscope dead and transparent larvae were found to be severely infected with endoparasitic stage of the protozoan (Ch. uncinata) that were more predominant in the haemocoel, head capsule, siphon region of the host larva. These protozoans were subsequently isolated from the cadavers from the disposable cups, then re-cultured and reared in the laboratory.
3.1 Chilodonella uncinata strains
The efficacy (% mortality values) of South India strain against An. stephensi, Cx. quinquefasciatus and Ae. aegypti are presented in Figure 1. The centrifuged strain at 2x104 cells/ml produced 96%, 72% and 32% mortality in An. stephensi, Cx. quinquefasciatus and Ae. aegypti respectively on day I after exposure; 100% mortality was observed in all the species on day 2 after exposure (Figure 1a). When the strain was not centrifuged, the least concentration 1x104 cells/mlproduced 77% and 85%, 82% and 92% mortality in An. stephensi and Cx. quinquefasciatus respectively on day 2 and day 3 after exposure (Figure 1b). The lowest concentration 3x104 cells/ml produced only 63% mortality on day 3 after exposure in Ae. aegypti (Figure 1c). Some promising results were noted with pre-monsoon and monsoon strain of Ch. uncinata NICDENTBPL 13106. With pre-monsoon strain at lower concentration (1x104 cells/ml) 100% mortality was observed on day 4 and day 6 after exposure against Cx. quinquefasciatus and Ae. aegypti respectively (Figures 2c, 2e). Still lower concentration 5x103 cells/ml produced 100% kill in An. stephensi on day 4 after exposure (Figure 2a). All the mosquito species were susceptible to Monsoon strain but at a much higher concentration. However, out of concentrations (1x105, 2x105, 4x105 and 8x105) cells/ml tested, 1x105 cells/ml produced 91% and 90% mortality in An. stephensi and Cx. quinquefasciatus after day 7 and day 8 respectively (Figures 2b, 2d); 87% mortality was recorded in Ae. aegypti at 2x105 cells/ml on day 12 after exposure (Figure 2f). Bioassay study undertaken at JMI with the centrifuged strain of Ch. uncinata BP 610 at the lowest dose of 1x103cells/ml against An. stephensi revealed 82% and 100% mortality on day 1 and day 4 respectively after exposure (Figure 3a). Whereas with un-centrifuged strain at the same concentration (1x103cells/ml) against the same species produced 16%, 76% and 88% mortality on day 2, 3 and 7 respectively (Figure 3b).


Figure 1 Laboratory efficacy percentage (%) mortality results for Ch. uncinata NICDENTBPL 13106 south India strain against various species of mosquito larvae at different concentration (cells/ml) (a) Culture strain centrifuged prior test (b),(c) Culture strain not centrifuged prior test [Experiments carried out at VCRC]


Figure 2 Laboratory efficacy percentage (%) mortality results for Ch. uncinata NICDENTBPL 13106 against mosquito larvae at different concentration (cells/ml). (a) Pre monsoon strain against 2nd instar An. stephensi larvae [AS] (b) Monsoon strain against 2nd instar An. stephensi larvae (c) Pre monsoon strain against 2nd instar Cx. quinquefasciatus larvae [CQ] (d) Monsoon strain against 2nd instar Cx. quinquefasciatus larvae (e) Pre monsoon strain against 1st instar Ae. aegypti larvae [AE] (f) Monsoon strain against 1st instar Ae. aegypti larvae [Experiments carried out at NICD]


Figure 3 Laboratory efficacy percentage (%) mortality results for Ch. uncinata BP 610 against 2nd instar An. stephensi larvae at different concentration (cells/ml) (a) Culture strain centrifuged prior test (b) Culture strain not centrifuged prior test [Experiments carried out at JMI]


3.2 Chilodonella uncinata formulations
Laboratory studies carried out at NICD with granular formulation of Ch. uncinata NICDENTBPL 13106 against different mosquito larvae are presented in Figure 4. The result revealed that least dose of 2g produced maximum mortality in An. stephensi (93%), in Cx. quinquefasciatus (90%) and in Ae. aegypti (88%) on day 5, day 7 and day 8 respectively.


Figure 4 Laboratory efficacy percentage (%) mortality results for Ch. uncinata NICDENTBPL 13106 formulation against various species of mosquito larvae at different dosages (in g) [Experiments carried out at NICD]


Laboratory bio-assay undertaken at ROSS with various dosages, e.g.: 0.25g, 0.5g, 1.0g, 2.0g, 4.0g, 8.0g and 16.0g of tea bag formulation against 1st/2nd instar Cx. quinquefasciatus larvae are presented in Table 1. On day 7 after exposure, while all larvae in control cup emerged into adults, not a single larva became pupa from any of the 7 experimental cups. Mortality ranging from 44% to 84% in larval instars was observed in experimental cups. Among the larvae those were still alive on day 7 after exposure, mostly were in third instar, some ware in 4th instar and few in 1st/2nd instar. The lowest dose (0.25 g) produced maximum mortality (84%) on 7th day after exposure in Cx. quinquefasciatus. In the next experiment also carried out at ROSS, 2nd instar An. stephensi larvae were exposed to tea bags at 0.25g, 0.5g, 1.0g, 2.0g/250ml and the results are presented in Figure 5 wherein 28%, 64% and 100% mortality was recorded with the lowest dose (0.25g) on 4, 6 and 8 day respectively after exposure.

Table 1 Laboratory bio-efficacy with Chilodonella uncinata BP 610 formulation wrapped in tea bags against 1st/2nd instar Culex quinquefasciatus larvae


Figure 5 Laboratory efficacy percentage (%) mortality results for Ch. uncinata BP 610 formulation wrapped in tea bags (in g) against 2nd instar An. stephensi larvae [Experiments carried out at ROSS]


At JMI, the least dose (0.1g) formulation in freshly prepared tea bag produced 46% and 92% mortality on day 1 and day 5 respectively after exposure in 2nd instar An. stephensi (Figure 6a). In Ae. aegypti, 12%, 44%, 68% and 100% mortality were recorded on day 1, day 4, day 5 and day 7 after exposure respectively with the least dose of 0.5 gm tea bag formulation (Figure 6b).


Figure 6 Laboratory efficacy percentage (%) mortality results for Ch. uncinata BP 610 formulation wrapped in tea bags at different doses (in g) against different mosquito larvae (a) 2nd instar An. stephensi larvae (b) 1st instar Ae. aegypti larvae [Experiments carried out at JMI]


4 Discussion
First instar larvae of Aedes aegypti and early second instar larvae of Anopheles stephensi and Culex quinquefasciatus were susceptible to different strains and formulations of Chilodonella uncinata. In general, all the strains and formulations produced better effect against An. stephensi than Cx. quinquefasciatus and Ae. aegypti.
Delayed development was noted in mosquito larvae exposed to Ch. uncinata formulation (Table 1). 100% emergence inhibition was recorded in Cx. quinquefasciatus for a period of 7 days, the time generally required for emergence of adult mosquitoes from 1st/2nd instar larvae. In general, the least dose, produce maximum mortality with minimum exposure as observed with monsoon strain in An. stephensi, Cx. quinquefasciatus and Ae. aegypti (Figures 2b, 2d, 2f); with the updated strain (BP 610) in An. stephensi (Figure 3b). Similar trend in efficacy was noted with granular formulation (NICDENTBPL 13106) in all the test species (Figure 4) and tea bag formulation in An. stephensi (Figures 5, 6a) and in Ae. aegypti (Figure 6b)

Our findings corroborate with that of (Canon, 2013) who reported that one of the advantages of using microbial pesticide is that they are often effective in very small quantities. Unlike other slow acting mycopathogen and entomopathogenic bacteria that can be used at a higher dose (Geetha and Balaraman, 1999) this particular slow acting entomopathogenic rotozoa can be used at the least dose.

The LT50 and LT90 (in day)s values for various strains and formulations against different mosquito species were represented as follows: The LT50 and LT90 values for pre-monsoon strain at concentration 5x103 cells/ml against An. stephensi larvae were 2.50 and 3.57 respectively and at 1x104 cells/ml against Ae. aegypti larvae were 3.35 and 5.37 respectively while for monsoon strain at 1x105 cells/ml against An. stephensi and Cx. quinquefasciatus were (4.71 and 6.84) and (6.86 and 8.00) respectively (Figure 7a), indicating thereby monsoon strain required 20x higher dose and longer exposure period to induce satisfactory mortality in An. stephensi larvae, one of the reason for this may be dilution of the pathogen during monsoon months; for updated BP 610 strain at 1x103 cells/ml against An. stephensi larvae these values were 2.95 and 6.04 respectively (Figure 7c); for NICDENTBPL 13106 formulation at 2g against An. stephensi, Cx. quinquefasciatus and Ae. aegypti larvae were (2.86 and 4.63), (4.20 and 6.80) and (5.08 and 7.83) respectively (Figure 7b), suggesting An. stephensi larvae were more sensitive to the pathogen followed by Cx. quinquefasciatus and Ae. aegypti larvae. Similarly, efficacy of the centrifuged strain was greatly increased resulting close to 100% mortality in An. stephensi in 24 hrs post exposure (Figure 1a). South India strain was more effective and comparatively higher doses were required when North India strain was used indicating thereby differences in virulence in geographical strains.  


Figure 7 LT50, LT90 values (in days) (a) Pre-monsoon and Monsoon strain  (b) NICDENTBPL 13106 Formulation (c) BP 610 strain and Tea bag formulation AE, Ae. aegypti; AS, An. stephensi; CQ, Cx. quinquefasciatus; M, Monsoon; P, Pre-monsoon; TB, Tea bag


As per LT50 and LT90 values, An. stephensi larvae were comparatively more sensitive (0.84 and 4.62) to freshly prepared tea bag formulation than Ae. aegypti larvae (3.93 and 6.27). Satisfactory efficacy was noted with LT50 and LT90 values 5.16 and 7.69 respectively against An. stephensi at 0.25 g tea bag even after 6 months of storage at room temperature. The updated strain (BP 610) was stable with hassle-free maintenance and its tea bag formulation was easy to store, transport and evaluate with a shelf life of >18 months. In general, formulations produced better effect in all the larval species than the protozoan strains. While the formulation using culture strain of Ch. uncinata NICDENTBPL 13106 (North India strain) was unable to withstand extreme cold (below >4°C) climatic situation in Delhi, formulation wrapped in tea bags prepared with updated culture strain of BP 610 at JMI was able to tolerate cold winter months at ordinary room temperature in Delhi with excellent recovery rate even after 18 months at ROSS. 
Earlier longitudinal field studies had already shownthat Ch. uncinata was capable of inducing natural check on the abundance of JE vector population in areas of Northern India with high prevalence of these protozoan parasites and the area so far remained free from JE though other parameters for an impending outbreak of JE remained the same with very high JE vector abundance in August, plenty of water birds and pigs in the area (Das, 2012). One such area was Safiabad, situated adjacent to Singhu border, North Municipal Corporation of Delhi, had been reporting large number of dengue cases every year including during current years’ big spurt in dengue cases wherein a total of 1919 cases, 12 deaths were reported in a week (13-19 Sept 2015) from Delhi. At the same time, during the same week (2015), >90% mortality was noted in Cx. tritaeniorhynchus mosquito larvae collected from paddy fields from Safiabad. Ae. aegypti larvae though being susceptible to Ch. uncinata infection were not accessible to this microbial control agent as breeding habitats of both the species are entirely different thus preventing Cx. tritaeniorhynchus females infected with Ch. uncinata from laying these protozoan parasites in potential breeding habitats of dengue vector including coolers, constructions sites, discarded containers in the area thereby not impacting population of dengue vector.
Unlike other entomopathogenic protozoan (L. clarki) that infect only container-breeding mosquito species such as Ae. sirensis (Washburn, 1995), Ch. uncinata has a wide host range in nature, viz.: Cx. tritaeniorhynchus, Cx. pseudovishnoi, Cx. (Lutzia) sp., Cx. (Culex) sp., An. stephensi mysoriensis and An. hyrcanus group (Das, 2003). This fact combined with the present findings of susceptibility of laboratory reared mosquito larvae, An. stephensi, Cx. quinquefasciatus and Ae. aegypti to all the strains and formulations of Ch. uncinata has provided great impetus to develop Ch. uncinata as a biological control agent for mosquitoes. Eight other observations noted earlier (Das, 2003; 2008; 2012) in the biology of Ch. uncinata that greatly enhances its biological control potential against mosquito vectors of human diseases: (i) the capacity of infected female mosquitoes to spread the parasite to new habitats via trans-ovarian transmission; (ii) can be produced in large scale using simple technology; (iii) tolerant to desiccation; (iv) robust and not sensitive to ultra violet radiation of sun and vagaries of agricultural pesticides; (v) facility to recycle in the environment, vi) free-swimming trophozoites of the parasite are the infective stage, and these actively seek out and infect larval hosts by drilling through the host cuticle; vii) not harmful to larvivorous fish, viii) female mosquitoes infected with Ch. uncinata are significantly less responsive toward a vertebrate host as compared to uninfected females. Some of these properties are shared by L. clarki in earlier finding of Egerter, Anderson and Washburn (1986) who suggested that L. clarki and related parasitic ciliates have much potential as biological control agents of mosquitoes. This entomopathogenic protozoan is a natural as well as indigenous, further studies on its biosafety test and small scale field trial are urgently required to develop these parasites as a simple, cost effective potential bio-larvicide that can be produced and stored easily.
5 Conclusions
Chilodonella uncinata is known to have control over mosquito vector of Japanese encephalitis in nature. The results of the present study indicate that a lower dose of this entomopathogenic protozoan formulation can be used as a potential biolarvicide to reduce vectors of malaria, dengue/Chikungunya and filariasis in the context of integrated vector management.
Authors’ contribution
BPD collected the protozoan infected mosquito larvae in field and isolated the protozoan in laboratory, developed various strains and formulations. She also performed bioassays at NICD and JMI and drafted early Manuscript. KD performed bioassays at ROSS, performed statistical analysis and participated in drafting the manuscript. KNP was responsible for developing the updated strain and its tea bag formulation. She also helped BPD in performing bioassay and maintained the updated strain till 2012 at JMI. RM is responsible for establishing Ch. uncinata strain from tea bag formulation prepared earlier and is maintaining Ch. uncinata culture at ROSS till date. He also assisted in bioassay evaluation of this Biolarvicide. SAH was instrumental in developing updated strain and tea bag formulation. PJ is responsible for original idea of the study, conceived the experimental design, and supervised the bioassay performed at VCRC.
We are grateful to the Department of Science and Technology, Government of India for a DST project catalysed and supported under its Utilization of Scientific Expertise of Retired Scientist Scheme to one of the authors (BPD). Co-operation and support of Amit Kumar, in-charge, Central Instrumentation Facility and Prof. L. Khan, Head, Department of Biosciences, Jamia Millia Islamia University (JMI), New Delhi for providing the laboratory facilities during the course of this study are gratefully acknowledged. Thanks are also due to Mudsser Azam, JMI for his assistance in preparation of tea bag formulation and Dr K. Balaraman, VCRC for his encouragements and guidance in this study. We thank G. Madhumita Das, Deputy Director General, Postal Service, Government of India for technically advising in preparation of the figures. The assistance rendered by Pratap Singh, N.S. Rawat, Karamvir Tussir and Late Rampat Tussir, Technical staff of NCDC are gratefully acknowledged.
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