J Arthropod-Borne Dis, December 2016, 10(4): 602–607 CD Patil et al.: Vorticella sp: Prospective … 602 http://jad.tums.ac.ir Published Online: October 04, 2016 Short Communication Vorticella sp: Prospective Mosquito Biocontrol Agent Chandrashekhar Devidas Patil, Chandrakant Prakash Narkhede, Rahul Khushal Suryawanshi, *Satish Vitthal Patil School of Life Sciences, North Maharashtra University, Jalgaon, Maharashtra, India (Received 14 Nov 2014; accepted 10 Nov 2015) Abstract Background: Considering the disadvantages of chemical insecticides, we aimed to evaluate Vorticella parasites for control of mosquito larvae of Anopheles stephensi and Aedes aegypti at different larval stages. Methods: Vorticella sp infected mosquito larvae were crushed in the 0.85% saline and homogenized well to get Vor- ticella in suspension. The effects of Vorticella sp infections on larval development were investigated by inoculating protozoan on different larval instars of An. stephensi and Ae. aegypti and observed under light microscope. Lethal time of the Vorticella infected larvae at different stages was calculated. Results: First and 2nd larval instars of both An. stephensi and Ae. aegypti did not show signs of infection by Vorti- cella sp., whereas 3rd instars of An. stephensi showed more Vorticella infection than those of Ae. aegypti. However, 4th larval instars of both mosquitoes were heavily infected with Vorticella parasite which was responsible for slug- gish movements of larvae and eventually death. Moreover, parasites (Vorticella spp) were responsible for more than 90% reduction in adult emergence for both infected An. stephensi and Ae. aegypti. Conclusion: This study provides insights for mosquito larvicidal action of surface parasite Vorticella on different larval stages of An. stephensi and Ae. Aegypti. It could be suggested as a potential candidate in mosquito biocontrol programs. Keywords: Anopheles stephensi, Aedes aegypti, Vorticella, Biocontrol Introduction Pathogenic agents of diseases like malaria, dengue fever, yellow fever and filariasis are mainly transmitted by mosquitoes and there- fore are responsible for human mortality and morbidity throughout the tropics (WHO 2004). As there is less development in drugs controlling pathogenic agents, mosquito vec- tor control has been recognized as an effec- tive tool for controlling the spread of these tropical diseases. Several synthetic chemical insecticides such as organophosphate, temephos, etc. have been used during the past several decades for the mosquito control. However, their use has been greatly impeded due to development of in- secticide resistant vectors (WHO 2013). Be- sides the use of chemical insecticides raised issues of environmental pollution which im- part harmful effects on non-target animals (Rao et al. 1995). Due to such reasons, there has been an increasing interest in developing biopesticides as a useful substitute to chemi- cal insecticides. Bacillus thuringiensis subsp israelensis (Bti) has been extensively used due to its specific- ity and high toxicity to a variety of mosquito larvae. Although Bti based strategies repre- sent an environmentally friendly approach to mosquito suppression with the potential for area-wide implementation, it is still lagging behind chemical insecticides, presumably due to its high cost and geometrical variation in Bti activity. In addition, the development of Bti resistance has made it difficult to control insect pests with Bti alone (Margot et al. 2011). Several insect control agents have been with- *Corresponding authors: Dr Satish Vitthal Patil, E-mail: satish.patil7@gmail.com J Arthropod-Borne Dis, December 2016, 10(4): 602–607 CD Patil et al.: Vorticella sp: Prospective … 603 http://jad.tums.ac.ir Published Online: October 04, 2016 drawn for economic or regulatory reasons re- sulting in greater selection pressure and more rapid resistance development to the remaining materials (Phillip et al. 2011). Thus, it is neces- sary to search new alternatives to develop prom- ising biological control against mosquitoes. Although various biocontrol measures are in vogue, their effective control is yet to be highlighted. In continuation of our previous studies (Chandrashekhar et al. 2011a, 2011b, Rahul et al. 2011, Suryawanshi et al. 2014, 2015) on searching of insecticidal agents for mosquito borne disease control, this study was conducted to isolate new insecticidal agents against mosquito larvae. Various sam- ples of dead mosquito larvae were collected from different mosquito breeding sites. We found extracellular protozoan parasite asso- ciated with these larvae. The parasite was identified as Vorticella, a genus of peritrich protozoan. It is one of the very first protists seen by Antony van Leeuwenhoek who ob- served it through the lens of his simple mi- croscope in the late seventeenth century. Vor- ticella has a bell shaped body with a cilia lined in oral cavity at one end and a long stalk on the other. Vorticella reproduces asexually by mitotic cell division (Noland and Finley 1931). They are sessile fresh water forms re- ported from different habitats like moist soil, mud and plant roots (Neumann and Marti- noia 2002). In this study we aimed for i) collection of the Vorticella sp in suspension form ii) col- lection of mosquito larvae from local breed- ing sites, iii) evaluation of Vorticella as a fu- ture biocontroling agent for mosquito larvae iv) calculation of lethal time of mosquito larvae for Vorticella suspension and v) to study the target larval selection by Vorticella sp. Materials and Methods Aedes aegypti and An. stephensi larvae (1st, 2nd, 3rd and 4th instar) were collected from nearby marshy places or stagnant ponds of Jalgaon area, at geographical coordinates of 21°2′54″N, 76°32′3″E, elevation 209 m and used as experimental mosquitoes. Key factor for primary identification was swimming be- havior of Aedes and Anopheles species (e.g. Anopheles larvae rests parallel while Aedes larvae attach to water surface with their body pointing downwards). From such samples, few larvae (5–10 depending on population size) were taken randomly for identification pur- pose. Identification of mosquito larvae was done by observing body parts like antenna, cephalon, mesothorax, metathorax and ab- dominal segments (Du Bose and Curtin 1965, Ramachandra 1984, Subbarao et al. 1988, Am- erasinghe 1990). For maintenance, the larvae were kept in plastic trays containing dechlorinated tap wa- ter. The experiments were carried out at 28± 2 °C and 75–85% relative humidity under 14:10 light and dark cycles. Larvae were fed with a diet of finely ground brewer's yeast and dog biscuits (3:1) (Chandrashekhar et al. 2011a, Suryawanshi et al. 2014). Larvae were observed under microscope (Labomade LB341), heavily infected larvae were identified by presence of attached para- sites all over the body surface, more popu- lated at the junction of all segments and sluggish movements of infected larvae. Five heavily infected mosquito larvae were se- lected. The larvae were further crushed in the 0.85% saline (5 ml) and homogenized well to prepare Vorticella suspension. The presence of Vorticella in the suspension was confirmed by observation under microscope. This sus- pension (5 ml) was inoculated into glass trays each containing the test 300 mosquito larvae (each instars) of An. stephensi or Ae. aegypti in one liter tap water containing glass tray and observations were made after each 24 h. Moreover, the effects of Vorticella sp in- fections on larval development were investi- gated by inoculating protozoan on the differ- ent larval instars of An. stephensi and Ae. ae- gypti. J Arthropod-Borne Dis, December 2016, 10(4): 602–607 CD Patil et al.: Vorticella sp: Prospective … 604 http://jad.tums.ac.ir Published Online: October 04, 2016 Similar experiment was done for observ- ing the preference of mosquito species by Vorticella by infecting Vorticella suspension to the mixture of both An. stephensi and Ae. aegypti larvae at their 4th larval instar. Mortality was Corrected using Abbott's formula (Abbott 1925). The dose–response data was subjected to probit regression anal- ysis (Finney 1971). Lethal time 50 (LT50) (the time at which 50% larvae died) of the larvae at different stages was calculated by probit analysis. Results After 24 h, around five to ten Vorticella sp stalk bodies could be observed on first seg- ment and 20–25 stalk bodies on last anal seg- ment of only 4th instar larvae of An. stephensi. Only a few stalked bodies of parasites were observed on 3rd instar larvae. While no any infection was observed on 1st and 2nd larval instars of An. stephensi. Similarly, Ae. ae- gypti larvae were not infected by protozoan up to 48 h and only few stalk bodies were observed on the first segment of 4th instars after 50 h after which infection was found to increase during the progression of life cycle. In contrast, the whole body surfaces of An. stephensi larvae were infected by the long stalked Vorticella sp up to 48 h (Fig. 1). Anopheles stephensi larvae did not showed abnormal changes in motility behavior for the first 48 h. However, the larvae became sluggish and then immotile after 48 h. During this stage, larvae showed movement of only upper mouthpart and siphon. When mixed lar- val populations of Anopheles and Aedes were inoculated with Vorticella the parasites preferred only Anopheles larvae for infec- tions. However, they heavily infect the Ae- des larvae after the death of Anopheles lar- vae. The effects of Vorticella sp infections on larval development were investigated by in- oculating the parasite into different larval instars of An. stephensi and Ae. aegypti. The 50% of 3rd and 4th instars population of An. stephensi larvae were killed after 72 h and 59 h, respectively (LT50). The 1st and 2nd in- stars larvae of both the mosquito species did not show any mortality up to72 h. The 3rd in- star of Ae. aegypti showed the infection but did not cause mortality. However, 4th instar larvae showed 50% mortality after 84 h. On- ly first segment of larvae was infected by parasites (Table 1). In adult immergence assay, out of the to- tal 300 larvae, 244 larvae of Ae. aegypti died within 84 h, 56 went to pupal stage and 23 adult mosquitoes were emerged. Upto 255 out of 300 An. stephensi larvae were killed, and 43 developed to pupal stage and 17 emerged to adult mosquitoes. The emergence rate of control An. stephensi and Ae. aegypti with- out Vorticella treatment was 73% and 76.1%, respectively (Table 2). Only 18.66% and 10% larvae of An. stephensi and Ae. aegypti attained pupal stages, respectively and died thereafter. Only 5.86% and 7.43% emerged as adult mosquitoes (Table 2). However, the emerged mosquitoes died within 4–5 days after their transfer and culture on the artifi- cial diet. Table 1. Effect of Vorticella sp infection on mortality of mosquito larvae Mosquito species Larval instars stage Infection LT50 (Hours) Aedes aegypti I II III IV Nil Nil D ++ -- -- -- 82.3±8.6 J Arthropod-Borne Dis, December 2016, 10(4): 602–607 CD Patil et al.: Vorticella sp: Prospective … 605 http://jad.tums.ac.ir Published Online: October 04, 2016 Anopheles stephensi I II III IV Nil Nil +++ ++++ -- -- 72±7 59±8.5 LT50= the time at which 50% larvae died D= detectable ++= infected only first segment of larvae and very few Vorticella parasites were observed +++= Vorticella parasites infected the whole larval body and many Vorticella parasites were observed ++++= very high infection throughout larval body and larvae be- came sluggish Table 2. Effect of Vorticella sp infection on emergence of adult mosquitoes Mosquito species Measure 4th instars larval mor- tality (number/ 300) Pupae emerged (number/300) Adults emerged (number/300) Aedes aegypti Controla Testb 39.3±4.04 244.67±15 260±15 56.6±9 228.3±12.6 22.3±5 Anopheles stephensi Controla Testb 29.6±4.5 255.7±10 271.6±17.5 43.6±6 219.3±17.7 17.6±5 aControl- larvae without Vorticella infection bTest- larvae with Vorticella infection Fig. 1. Infection of parasite (Vorticella spp) to the 4th larval instars of Anopheles mosquito (10X magnification) Discussion Vorticella spp. are largely known as bac- terial feeders and have also been reported to parasitize nematodes with the help of their integument, in Rana pipiens and Rana syl- vatica tadpoles it cause tegumentary im- pairment while in fresh water prawns Acro- brachiumrosen bergii Vorticella sp infect the eye stalk, antenna, uropod and egg masses (Tonguthai 1997). Ciliates and Vorticella sp have been found occasionally infecting mos- quito larvae (Muspratt 1945, Micks 1950, 1955, Schober 1967). Vorticellids of Vorticel- la can cause infection in mosquito species at risk (Micks 1950). In the present study, the effects of Vorti- cella sp on all larval instars of An. stephensi Table 1. Continued… J Arthropod-Borne Dis, December 2016, 10(4): 602–607 CD Patil et al.: Vorticella sp: Prospective … 606 http://jad.tums.ac.ir Published Online: October 04, 2016 and Ae. aegypti were investigated. The study on life cycle of the infected An. stephensi and Ae. aegypti larvae has revealed the inhi- bition of larval growth, development and adult emergence of both the test mosquito species. Micks (1950) reported Vorticella infection in Anopheles and death of 7000 infected larvae. However, the mechanism behind this inhibi- tion is still unknown since more than 74 years of first report. The foot of the stalk at- taches to a substrate, such as a rock, plant, or even an aquatic animal. The stalks of these organisms are capable of contracting at astounding speeds. The mechanism by which the adhesive pad of V. convallaria adheres to so many different substrates is also unknown. The organism secrets a some sort of “glue”, probably a biopolymer. It may be possible that organism secretes some biochemical sub- stances with these glue to fix itself on sub- strate and those substances may damaging some surface sensory system or causing pore formation in larval body. Either it may also be possible that the metabolic and secretary products of Vorticella are toxic to the mos- quito larvae, polluting its natural environment. As per Schober (1967) this association is benefited by Vorticella as it may be using host as a transportation vehicle to spread in new environment, while as per Mick (1955) the lar- val death may be apparently due to the inabil- ity of the infected larvae to remain on the wa- ter surface, thereby interfering with respiration. Previously it was observed that Vorticella infection was found only in Anopheles and it did not show either infection or mortalities in Aedes aegypti. In the present study, alt- hough Vorticella sp has first preference to Anopheles but it can attack other mosquito species like Ae. aegypti. The observations from this study can be important to design strategy to control of mosquito larvae. Fur- ther studies on non-target organisms and persistence in different environment will be needed for use of Vorticella sp as potential biocontrol agent in the mosquito larval habitat. 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