Tropical Biomedicine 31(2): 207–214 (2014)
Coexistence of Philodina roseola (Rotifera: Bdelloidea) with larvae of Aedes aegypti in India Muniaraj, M.* and Sathish Babu, R. Centre for Research in Medical Entomology (Indian Council of Medical Research), No. 4, Sarojini Street, Chinna Chokkikulam, Madurai – 625002, India *Corresponding author email:
[email protected] Received 30 August 2013; received in revised form 25 October 2013; accepted 29 October 2013
Abstract. The vector mosquitoes, Aedes aegypti and Aedes albopictus of dengue and Chikungunya fever are closely associated with human habitations and adapted to feed on human blood. They undergo larval and pupal development in natural and artificial freshwater collections in the urban and peri-urban environment. Although reports are available about the feeding behaviour of the thriving mosquito larvae, much information is still required to understand the successful survival of Aedes mosquitoes in small and temporary water collections. This study was undertaken to determine the co-existence and prevalence of Philodina roseola and other Bdelloid rotifers in the container habitats of Ae. aegypti mosquitoes. The investigation was conducted in 43 villages which belong to four districts in South India, affected by the epidemic of either dengue or Chikungunya fever. A total of 2093 houses and 12980 containers were examined for Aedes breeding and those containers with Aedes larvae were chosen for further investigation. The investigation showed that, the P. roseola was found associated in 502 (98.2%) containers, P. roseola along with other Philodina sp. in 126 containers (25%) and P. roseola along with other Philodina sp. and other Bdelloid rotifers found in 93 containers (19%). Since the members of the genus Philodina can survive desiccation, reproduce by parthenogenesis, can be transported by wind easily and more importantly, it can incorporate the genome of other organisms including viruses, understanding the coexistence and relationship of Philodina sp. with Aedes larvae would be helpful in the control of Aedes breeding and the control measures can be designed keeping the association of Bdelloids with Aedes in mind.
associated with human habitations (Gubler, 1998; Rigau-Perez et al., 1998). They have adapted to feed on human blood and undergo larval and pupal development in natural (e.g. rock pools, tree holes, leaf axils) and artificial (e.g. water tanks, blocked drains, decorative pots and discarded tyres and food/beverage containers) freshwater collections in the urban and peri-urban environment (Ramasamy et al., 2011). The thriving mosquito larvae feeds on microorganisms such as bacteria, cyanobacteria, fungi, protozoans, unicellular algae and diatoms (Thiery et al., 1991; Pumpuni et al., 1996; Donmez et al., 1999; Ponnusamy et al., 2008). However, more information is required to
INTRODUCTION Dengue and Chikungunya fever are the most important human viral diseases transmitted by mosquito vectors, Aedes aegypti and to a lesser extent, Aedes albopictus. Annually there are an estimated 50–100 million cases of dengue fever (DF), and 250 000 to 500 000 cases of dengue haemorrhagic fever (DHF) with 20 000–25 000 deaths occuring around the world (Gubler, 1997; Chaturvedi & Shrivastava, 2004; Staples et al., 2009). Chikungunya fever is an acute febrile illness caused by an arthropod-borne alphavirus, Chikungunya virus. The vector mosquitoes, Ae. aegypti and Ae. albopictus are closely
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understand the successful survival of Aedes mosquitoes in small and temporary water collections. Placed in the class Bdelloidea (phylum Rotifera), which is Greek for leech, members of the genus Philodina include some of the smallest metazoans ranging from 0.1 to 1 mm in length and most are microscopic, live in freshwater, and free swimming. With two anterior rotating wheel organs referred to as coronas, Philodina can move like leeches or inchworms, extending and contracting as they crawl over aquatic plants and detritus. They can contract, extend their coronas and create water current to bring the prey towards rostrum (Melone & Ricci, 1995). The class Bdelloidea, consists entirely of females reproducing by apomixis, in which diploid eggs produced by mitotic division develop parthogenetically into females (Welch & Meselson, 2000; Birky, 2004) and successfully surviving for past 80 million years (Birky, 2004). The presence of Bdelloid rotifer, Philodina in the breeding containers of Aedes mosquitoes is considered to be highly significant due to the following three reasons. Firstly the Philodina sp. feeds on smaller bacteria, algae and prozoans or organic materials and increases in size and number and can serve as food for mosquito larvae (Muniaraj et al., 2012). Secondly anhydrobiosis; Philodina can live virtually without water in an abiotic state for long periods of even several decades and can resume activity once the container is filled with water (Lapinski & Tunnacliffe, 2003). Thirdly, under dried state, rotifer Philodina can be transported by wind to a relatively larger distance of several hundred kilometres and resume life once it falls on any water body (Orstan, 1998; Van Doninck et al., 2009). Hence if a dried container is filled with water, the already existing dehydrated form or transported by wind, the anhydrobiotic Philodina sp. could be the first organisms to resume (Lapinski & Tunnacliffe, 2003) and act as an immediate source of food for the larvae. The bdelloids are extremely resistant to ionizing radiation and has capability of repairing damaged DNA very fast made them to survive in any harsh environment and recover without any loss (Gladyshev &
Meselson, 2008). This means uninterrupted availability of food source for Aedes larvae with possible long evolutionary relationship. A recent evidence suggests that Philodina sp. acquired genes from other organisms by horizontal gene transfer (HGT)( Boschetti, 2012) and another study reported the presence of reterovirus-like genetic materials in the transposons of Bdelloid rotifers (Gladyshev et al., 2007) suggesting the possibility of Bdelloids to be the hiding source for dengue or other arboviruses. Although the presence of Philodian sp. in the breeding container of Aedes was reported (Muniaraj et al., 2012), its co-existence, prevalence and distribution have not been investigated. The main objective of this report was to investigate the coexistence of Bdelloid rotifer, P. roseola with larvae of Ae. aegypti as prey and predator, the prevalence and distribution of both, in the breeding containers.
MATERIALS AND METHODS Study site The study sites were selected on the basis of prevailing epidemic of dengue/ Chikungunya fever. The criteria for selection of sites were based on the high incidence of confirmed cases of dengue/ chikungunya fever recorded by the health department of affected States. Every house in the affected village was visited to ascertain the breeding potential. Fourteen villages in Ramanathapuram district (Tamil Nadu) viz. Muthuramalingapuram, Natarajapuram, Chinnapallam, Puthu Road, Kenthamathaparvatham, Thekkuvadi, Thiruvadani, Thondi, R.S. Mangalam, Devipattinam, Kuthukkalvalasai, Puthumayakkulam, Pullanthai and Keelakarai; 16 villages in Dharmapuri district (Tamil Nadu) viz. Indur, Nagadesampatti, Tekkal Naikkanpatti, Kadathur, B. Mallapuram, Dasarahalli-Morappur, Annal Nagar-Morappur, Sekkodi, Murukkampatty, Chinna Gumanur, Jakkasamudram, K o t t u m a r a n a h a l l i - P e r i y a m p a t t y, Kamalapuram-Periyampatty, LaligamAppanahalli kombai, Sivadi-Palayampudur 208
and Keel & Mel Esalpatty-Thoppur; 6 villages in Warangal district (Andhra Pradesh) viz. Warangal city, Venkatapuram, Kowkonda, Issipet, Sudanapally, and Seethanagaram; 7 villages in Adilabad district (Andhra Pradesh) viz. Ramnagar, Chunnampattivada, Ashok nagar, Gollavada, Kasipet, Kurumapally and Arunakanagar. Both Tamil Nadu and Andhra Pradesh are the States located in the southern part of India (Fig. 1). A series of entomological investigations was carried out to find out the breeding habitats of Aedes mosquitoes and distribution of Bdelloid rotifer, Philodina, during the Chikungunya fever outbreak of 2009 and 2010 in Ramanathapuram district and dengue fever outbreak of 2010 in Dharmapuri district and 2012 in Warangal and Adilabad district. Four rounds of investigation, covering 14 villages in Ramanathapuram, a single round of investigation covering 16 villages in Dharmapuri, 6 villages in Warangal and 7 villages in Adilabad districts were carried out.
Search for the breeding habitats of Aedes and Bdelloid rotifers A total of 2 093 houses and 12 980 containers were examined for Ae. aegypti breeding and the samples from the breeding containers were selected for further investigation for the presence of P. roseola and other Bdelloid rotifers. The water samples were collected by slightly scrubbing the inner wall of the container for any attached organic particles or Bdelloid rotifers. Water samples from the breeding containers were carefully collected in sterile plastic bottles and brought to the laboratory to find out the presence of Bdelloid rotifers. The samples were observed under stereomicroscope by placing 100-1 000 µl in a wide cavity slide with a dark background. If there are no organisms observed, then the samples were centrifuged at 1 500 rpm for 5 minutes to concentrate the organisms. If still no organisms are visible, then the samples were dispensed in 6 or 8 centrifugal tubes and centrifuged. Then the concentrated
Figure 1. Map of India showing study districts, 1. Ramanathapuram, 2. Dharmapuri, 3. Warangal, 4. Adilabad
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samples were collected in one tube by removing the supernatant and then the collected tube was centrifuged again and the supernatant was discarded. After the confirmation in the stereomicroscope, around 100 µl was collected from the cavity slides and placed on a clean glass slide with glass cover-slip placed on to avoid evaporation and observed under compound microscope for the morphological features through 10x magnification. Even then if no rotifer was found, then the sample was considered to be negative for Bdelloid rotifers. Although some other rotifers which are morphologically different from the Bdelloids, were noted in some of the containers, we concentrated only on the presence of Bdelloids. Aedes aegypti immature collection was done using a standard dipper (300 ml with 9 cm diameter) in larger containers. In smaller containers, the sampling was performed using a dipper of 150 ml capacity with 6.5 cm diameter or with glass pipettes connected with rubber bulb. Container breeding in peridomestic and intradomestic areas were qualitatively and quantitatively assessed. The immature of both larvae and pupae collected were scored and subsequently allowed to hatch for identification of the mosquito species.
both sets and recorded. This experiment was carried out for seven days and the results were plotted on a graph..
RESULTS A total of 407 (19.4%) houses and 511 (3.9%) containers were found positive for Ae. aegypti breeding out of 2 093 houses and 12 980 containers examined. Aedes albopictus had not been found in any of the containers. Out of 511 containers positive for Ae. aegypti, we were able to identify the presence of P. roseola in 502 (98.2%) containers, P. roseola along with other Philodina sp. in 126 containers (25%) and P. roseola along with other Philodina sp. and other Bdelloid rotifers found in 93 containers (19%) (Fig. 2). The major containers for the breeding of A. aegypti were discarded containers (28.4%) followed by cement cistern (25.2%), plastic containers (15.4%), metal containers (6.4%), mud pot (5.3%), flower pot (4.7%), refrigerator tray (3.9%), cement tank (3.3%), grinding stone (2.1%), flower vase (1.6%), tyre (1.6%), coconut shell and air-cooler (0.6% each), sump (0.4%), plastic and metal drum (0.2% each) and no breeding was observed in both over head tank and well. The distribution of P. roseola and other Bdelloids was also following the similar pattern with some
Experiment to prove the role of P. roseola as larval food for Aedes larvae In a triplicate set up of 500 ml capacity beaker, 250 ml natural mineral water (Aava, India) was taken in each. Then the laboratory cultured active P. roseola culture was added with final load of 2x102/ml. With this, 25 fourth instar larvae of Ae. aegypti was added and incubated in room temperature. Next day, the larvae were removed; the number of P. roseola was counted and a fresh set of larvae was added and incubated again. The alimentary tract of the removed larvae were dissected and examined under compound microscope for the presence intact Philodina or the Jaw of mastax - an organ with chitinous plates called trophy (jaws) that chops food and pushes it into the esophagus. Another triplicate set of beakers with 250 ml natural mineral water was inoculated with P. roseola to give final load of 2x102/ml and kept as control. Number of Philodina was counted in
Figure 2. Distribution of P. roseola and other Bdelloids in the breeding containers of Ae. aegypti mosquitoes
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difference. The major habitat for Bdelloid rotifer was discarded containers (28.9%) followed by cement cistern (25.7%), plastic containers (15.5%), metal containers (6.6%), mud pot (5.4%), flower pot (4.8%), refrigerator tray (4%), cement tank (3%), grinding stone (2%), flower vase (1.6%), tyre (1.2%), aircooler (0.6%), sump (0.4%), plastic and metal drum (0.2% each) and no P. roseola was observed in samples from coconut shell (Fig. 3 & 4). Among the Bdelloid rotifers, P. roseola was alone found in 56% of containers followed by coexistence of P. roseola with other Philodina sp. in 25% of containers and in 19% of containers, P. roseola coexisted with other Philodina sp. and other Bdelloids. Among the Ae. aegypti breeding containers, 100% positivity for Philodina with or without other Bdelloids was observed in discarded containers (145/145), cement cistern (129/ 129), metal container (33/33), mud pot (27/ 27), flower pot (24/24), refrigerator tray (20/ 20), flower vase (8/8), air-cooler (3/3), sump (2/2), plastic drum (1/1) and metal drum (1/1), followed by plastic container (78/79), cement tank (15/17), grinding stone (10/11), tyre (6/8). In the experiment carried out for
finding the nutritional role of Philodina, the number of P. roseola was found constantly decreased from 195 on 1st day to 42 on 7th day. In contrast, the control showed only a slight decline in the number of Philodina from 198 of 1st day to 162 of 6th day (Fig. 5). Although the intact Philodina was not found in the contents of the alimentary tracts of the dissected 175 Aedes larvae, the jaw of mastax was found in 23 (13.1%). Since, no food is added for the Philodina, in control too, their number declined, but slowly.
DISCUSSION The 98.2% prevalence of P. roseola, 25% of other Philodina sp. and 19% of other Bdelloids in the Ae. aegypti breeding containers strongly reveal the association of Bdelloids particularly, P. roseola in the existence of Ae. aegypti mosquitoes. The constant declining of P. roseola and the presence of Jaw of mastax in the gut contents of Aedes larvae clearly proves the nutritional role of P. roseola for the development of Aedes larvae. It seems that the discarded containers and
Figure 3. The percentage positivity of containers for Philodina sp.
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Figure 4. Number of containers positive for P. roseola, other Philodina sp. and other Bdelloid rotifers in Ae. aegypti breeding containers
Figure 5. Reduction in the number of Philodina in the containers with Aedes larvae indicates its role as larval food. The control shows slow declining in the number of Philodina which was mainly due to lack of nutrients that led its normal death
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cement cisterns and plastic containers provide the required organic or photosynthetic microbial source for the growth of P. roseola that in turn supported the breeding of Aedes mosquitoes. The complete absence of Philodina or other rotifer in coconut shell might be due to the composition of water in the disposed coconut shells that have not supported the growth of P. roseola but sufficient enough to support the growth of Aedes. The size of the container is having negative association with the breeding of Aedes, which generally prefer smaller containers rather than larger tank/ drum /sump. By virtue of its characters such as anhydrobiosis (Lapinski & Tunnacliffe, 2003), resistance to radiation, desiccation (Tunnacliffe, 2009), easily transported by wind (Orstan, 1998; Van Doninck et al., 2009), reproduce parthogenetically (Welch & Meselson, 2000; Birky, 2004), bio-mass magnifier (Muniaraj et al., 2012), horizontal gene transfer (Gladyshev et al., 2008) etc. the Bdelloid rotifers, particularly, P. roseola seems to be highly essential for the breeding of Ae. aegypti and perhaps other similar mosquitoes such as Ae. albopictus. In this study, Ae. albopictus was not recovered from the breeding habitats. It has been reported that, Ae. albopictus is uncommon in the breeding habitats of areas other than the Kerala State, where, Ae. albopictus adapted to live in the container habitats (Alex Eapen et al., 2010). The universal distribution of P. roseola and other Bdelloids probably reveal its hidden but most important role in the flash emergence of Aedes larvae after every rainfall in the tropical areas where several mosquito-borne infections are common. This association is also important since, P. roseola and other Bdelloid rotifers can engulf or incorporate foreign DNA with its genome by horizontal gene transfer and seem to be acting as a hiding source for the arboviruses and act as intermediate agent for the transmission to the vector mosquitoes. It is concluded that the versatile organisms like P. roseola and other Bdeolloids coexist with Aedes larvae in the breeding containers. The Bdelloids seems to be the main food source of growing larvae and possibly leading a prey-predator
relationship and is responsible for the successful survival of Aedes in small, temporary water collections. Acknowledgments. Dr. N. Arunachalam is thankfully remembered for his technical advice to carry out this study. The technical assistance of S. Victor Jerold Leo, A. Selvam, K. Manimaran, J. Balasubramanian, and A. Govindasamy is greatly acknowledged. The help rendered by Dr. D. Narahari, Hyderabad, AP and Dr. Neelkanteshwar Rao, Mancheral, AP in conducting survey at Warangal and Adilabad district is thankfully remembered.
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