Evaluating the carnivorous efficacy of Utricularia aurea (Lamiales: Lentibulariaceae) on the larval stages of Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti (Diptera: Culicidae)

Abstract

The emergence of insecticide resistance in mosquitoes necessitates the exploration and validation of sustainable biological strategies for controlling mosquitoes in their natural habitats. We assessed the predatory effect of Utricularia aurea Lour (Lamiales: Lentibulariaceae), an aquatic carnivorous plant found in the Indian subcontinent, Japan, and Australia, on 4 instars of Anopheles stephensi Liston, Culex quinquefasciatus Say, and Aedes aegypti Linn (Diptera: Culicidae), in the laboratory and field settings. In the laboratory setting, predation of larvae by U. aurea was highest during the first hour when it predated 45%, 61%, and 58% of first instars of An. stephensi, Cx. quinquefasciatus, and, Ae. aegypti, respectively, and, within 12 h, U. aurea preyed upon ~95% of the first, second, and third instars of the 3 mosquito species, ~80% of the fourth instars of An. stephensi and Ae. aegypti, and ~60% of fourth instars of Cx. quinquefasciatus. The predatory effect of U. aurea varied with mosquito species and instar. Broadly, predation risk declined with the increase of the instar size. In the field setting, at the end of 16 days, U. aurea predated 76% and 71% of the immature An. stephensi and Ae. aegypti, respectively. Our findings suggest U. aurea can be utilized as a potential biocontrol agent for controlling mosquito larvae in natural habitats; however, the current claim warrants additional investigations in a variety of natural habitats.

Introduction

Mosquito-borne diseases, such as malaria, dengue, chikungunya, Japanese encephalitis, and filariasis, cause most of the febrile illnesses in India (Robinson et al. 2018). Malaria is primarily spread by Anopheles stephensi Liston (in western and southern India), Anopheles fluviatilis James and Anopheles culicifacies Giles (in central and eastern India), Anopheles baimaii Sallum & Peyton, and Anopheles minimus Theobald (in north-eastern India) (Subbarao et al. 2019). In India, dengue and chikungunya are primarily spread by Aedes aegypti Linn. (Mutheneni et al. 2017). Filariasis and Japanese encephalitis are transmitted by species belonging to Aedes, Culex, and Mansonia genera. These mosquito vectors are present in all types of habitats ranging from urban-polluted small water bodies to large brackish and freshwater bodies, making their control very difficult (Barraud 1934, Nagpal et al. 2005).

Integrated vector control management in India recommends using temephos (an organophosphate insecticide) for controlling mosquito larvae, and pyrethrum spray and malathion fogging for controlling adult mosquitoes, especially during their breeding season. However, the emergence of insecticide resistance in mosquitoes and the harmful effects of the insecticides on nontargeted organisms such as silkworm moths, honey bees, notonectid bugs, fishes, etc. warrants exploration, development, and validation of alternative and sustainable biological strategies for controlling mosquitoes in their natural environments (Kumar et al. 1994, Raghavendra et al. 2017, Bharati and Saha 2018, Rai et al. 2019).

Sustainable biological methods to control mosquito larvae include larvivorous fish, odonates, copepods, tadpoles, and aquatic carnivorous plants (Kumar et al. 1994, 1996, 1999, Imbahale et al. 2011). A promising, but understudied biological control strategy for controlling mosquito larvae is the use of Utricularia aurea Lour (Lamiales: Lentibulariaceae), an aquatic carnivorous plant also known as bladderwort. It is an oligotrophic plant that partially derives nutrition from its prey (Poppinga et al. 2015) by capturing a wide range of organisms belonging to Tardigrada, Nematoda, Gastropoda, Acaridae, Rotifera, Ciliata, Arthopoda, and Crustaceae (Darwin and Darwin 1888, Matheson 1930, Andrikovics et al. 1988, Mette et al. 2000, Harms 2002, Sanabria—Aranda et al. 2006, Gordon and Pacheco 2007, Guiral and Rougier 2007, Alkhalaf et al. 2009, Kurbatova and Yershov 2009). The suction trap of Utricularia is the fastest known trap among all carnivorous plants and can capture prey within 500 µm of the trapdoor (Vincent et al. 2011). The captured prey dies due to anoxia and is digested by a set of hydrolases and low-pH environments (Adamec et al. 2010). Although the predation of mosquito larvae by Utricularia was reported in the early 1930s, the use of Utricularia for controlling mosquito larvae is relatively underexplored. Under laboratory conditions within 2 days, Utricularia reflexa preyed upon 100% of An. gambiae, the African malaria vector (Ogwal-Okeng et al. 2011, Ogwal-okeng et al. 2013). Similarly, Utricularia macrorhiza, widely distributed in North America, was shown to kill 100% of Ae. aegypti and 95% of Ae. albopictus larvae within 5 days (Couret et al. 2020). Here, we report for the first time the predatory ability of U. aurea that is widely distributed in the Indian subcontinent, Japan, and Australia (Taylor 1989, Janarthanam and Henry 1992, Rutishauser 1993, Sanjeet Kumar et al. 2019) against the larvae of Ae. aegypti, An. stephensi, and Cx. quinquefasciatus over time in laboratory and field settings.

Materials and Methods

Collection of the Mosquito Larvae and Determination of Their Size

The mosquito larvae were obtained from the insectary of the National Institute of Malaria Research, Goa. The body length of larvae belonging to the first, second, third, and fourth instars of Ae. aegypti, An. stephensi, and Cx. quinquefasciatus was measured from preclypeus to the eighth abdominal segment using a microscale and stereomicroscope (Wild).

Collection and Identification of U. aurea

Utricularia aurea shoots of 10 cm length were collected from the lake at Band Bhat area, St. Cruz, Goa, India (15°28ʹ11.4″N 73°50ʹ26.2″E). The shoots were transported to the laboratory in a 5-liter bucket filled with the same reservoir water. The plants were identified using the methods described by Taylor (1989) and Janarthanam and Henry (1992).

Predation of the Larvae by U. aurea in Laboratory Mesocosms

The 10-cm shoots of U. aurea containing ~100–110 traps were added to 462-ml capacity plastic cups (radius × height: 4.4 × 7.6 cm) containing 250 ml of RO water. The 10-cm U. aurea shoot covered approximately 1/4th volume of the plastic cup. Twenty-five larvae of each instar stage of Ae. aegypti, An. stephensi, and Cx. quinquefasciatus were introduced into separate cups, in quadruplicates, containing the shoot of U. aurea (the ratio between the trap and larvae was 4:1). The experimental controls were larvae without the U. aurea shoot. The larvae were fed once with ~5 mg of fish food (Tetramin). Hourly capturing activity of the U. aurea bladder was recorded from 0 to 12 h. The laboratory experiments were carried out at a temperature of 27 ± 2 °C and relative humidity of 65 ± 5%.

Predation of the Immature Mosquitos by U. aurea in Field Mesocosms

In India, to prevent water loss from (or drying of) the concrete slabs of newly constructed buildings, water is allowed to stay over the surface of slabs continuously for several days; this method of keeping newly constructed concrete slabs inundated with water is referred to as curing, and the stagnant water is called as curing water. Furthermore, to effectively hold the curing water, several small water bodies (sites) are created, using a sand–cement mixture (or soil), on top of the newly constructed concrete slabs. In this study, a newly constructed concrete slab flooded with curing water and located in the Ponda area, Goa, India, was surveyed for the presence of immature (larvae and pupae) An. stephensi and Ae. aegypti and was chosen for the field study. This relatively large concrete slab had several small water bodies (sites) of equal size to hold the curing water. Each water body (site) was 1 m in length, 1.2 m in breadth, and 0.17 m in depth. Six such small water bodies (sites) with curing waters were considered mesocosms (mosquito developmental sites) for the field experiment and were maintained as either control or treatment mesocosms. Specifically, while the control mesocosms did not contain U. aurea shoots, the treatment mesocosms contained U. aurea shoots. Ten large shoots (~30 cm long) of U. aurea were introduced into each of the treatment mesocosms; 2 shoots per corner (4 corners), and 2 shoots in the center. Utricularia aurea covered about 1/20th of the volume of these mosquito developmental sites. Using the dipping method as previously described (WHO 2013) (5 dips were taken, one from each corner and one from the center every day for 16 days), the immature stages (larvae and pupae) of An. stephensi and Ae. aegypti present in the 3 control mesocosms and treatment mesocosms were collected, and subsequently, their numbers were recorded. Finally, the percent reduction of immature An. stephensi and Ae. aegypti in the treatment mesocosms when compared with the control mesocosms was calculated daily for 16 days using Mulla’s formula (Mulla et al. 1971).

Statistical Analyses

Larval length data are presented as means ± 1 standard deviation. The lengths of the 3 mosquito species at each instar stage were compared using a full-factorial, 2-way ANOVA with species and instar as fixed factors. Tukey’s post hoc tests were used to examine pairwise comparisons between species and instar lengths.

Cox proportional hazards regression analysis (Cox 1972) was used to evaluate the predatory effect of U. aurea on each instar and mosquito species. Larvae remaining alive at the end of a trial (i.e., at 12 h) were right censored. Species and instar were used as fixed factors. Mesocosm replicates were included in the Cox proportional hazards analysis as a random factor to account for pseudoreplication; 4 replicate mesocosms housed 25 larvae for each mosquito species and instar.

All statistical analyses were performed using the R statistical package (R Core Team 2023). Cox proportional hazards regression was performed using the “coxph” function in the “survival” package (Therneau 2023).

Results

Overall, the lengths of different instars of An. stephensi were smaller than those of Cx. quinquefasciatus, which in turn were smaller than those of Ae. aegypti, and length increased with instar (Fig. 1; species effect: F_{2, 48} = 289.33, P < 0.0001; instar effect: F_3,48 = 843.95, P < 0.0001; interaction effect: F_6,48 = 16.99, P < 0.0001). Except for the comparison between lengths of the first instar of Cx. quinquefasciatus and An. stephensi (Tukey HSD: P = 0.48) and between the lengths of the second instar of Cx. quinquefasciatus and Ae. aegypti (Tukey HSD: P = 0.87), the lengths of all instar stages significantly differed among the 3 mosquito species (Tukey HSD: P < 0.05).

Utricularia aurea Lour, either completely or partially, captured the 4 instars of Cx. quinquefasciatus, An. stephensi, and Ae. aegypti (Fig. 2). Specifically, the bladders of U. aurea completely engulfed the smaller mosquito larvae (Fig. 2A, B, E, F, G, and I) and partially engulfed the bigger mosquito larvae (Fig. 2C, D, H, J, K, and L).

In a 12-h laboratory predation experiment, U. aurea, at different rates, captured individuals of all 4 instars from each mosquito species (Fig. 3). No larval deaths occurred in the controls (laboratory mesocosms with the larvae and without U. aurea). In the laboratory, predation of larvae by U. aurea was highest during the first hour when it predated 58%, 45%, 58%, and 61% of first instars of An. stephensi, Ae. aegypti, and Cx. quinquefasciatus, respectively; and within 12 h, U. aurea preyed upon ~95% of the first, second, and third instars of the 3 mosquito species, ~80% of the fourth instars of An. stephensi and Ae. aegypti, and ~60% of fourth instars of Cx. quinquefasciatus. Cox proportional hazards regression was used to test whether U. aurea predation varied by species and instar. The main effects of mosquito species (Cox regression, χ² = 24.87, P < 0.0001, df = 2) and instar (Cox regression, χ² = 240.23, P < 0.0001, df = 3) impacted larval survival in mesocosms. Furthermore, there was a statistically significant interaction between species and instar (Cox regression, χ² = 152.53, P < 0.0001, df = 6), suggesting that larval predation by U. aurea varies by species and instar.

Across all instars, predation by U. aurea was reduced for An. stephensi compared to Ae. aegypti and Cx. quinquefasciatus, whereas predation was similar between Ae. aegypti and Cx. quinquefasciatus (Figs. 3 and 4). Across all mosquito species, predation was highest for second-instar larvae, second highest for first-instar larvae, and decreased with instar size for third- and fourth-instar larvae (Figs. 3 and 4).

The predatory effect of U. aurea on the immature (larvae and pupae) Ae. aegypti and An. stephensi in their natural developmental sites was assessed (Fig. 5). The relative numbers of larvae of both mosquito species were reduced throughout the trials, and at the end of 16 days, U. aurea reduced the density of immature Ae. aegypti and An. stephensi by 76% and 71%, respectively.

Discussion

We assessed the predatory ability of U. aurea on the different larval stages of Ae. aegypti, An. stephensi, and Cx. quinquefasciatus in laboratory and field settings. Utricularia aurea predated the mosquito larvae, and this observation is consistent with the earlier reports on the predation of mosquito larvae by other species of Utricularia such as U. reflexa (Africa) (Ogwal-Okeng et al. 2011, Ogwal-okeng et al. 2013) and U. macrorhiza (North America) (Couret et al. 2020). Previous studies have reported the number of mosquito larvae predated by Utricularia spp. each day for 5 days (Ogwal-okeng et al. 2013). Here, we report the hourly larval predatory rates of U. aurea for 12 h. Utricularia aurea predated ~95% of the first, second, and third instars of the 3 mosquito species, ~80% of the fourth instars of Ae. aegypti and An. stephensi, and ~60% of fourth instars of Cx. quinquefasciatus, whereas the predatory activity of U. macrorhiza on first to third instars of Ae. Aegypti and Ae. Albopictus over 24 h was 71.5% and 81.5%, respectively (Couret et al. 2020). The increased predatory activity of U. aurea could be due to the initiation of the experiments within 24 h of collecting the plants from their natural environment, while experiments with U. macrorhiza were carried out after 30 days of acclimatization in the laboratory (Couret et al. 2020). The laboratory acclimation to containers without nutrient inflows is probably analogous to what would happen to these plants when they are introduced to the curing water sites. Laboratory acclimatization (reduces the trap size) and nutrient availability are key determinants of Utricularia’s predatory ability (Twinn 1931, Friday 1991, Knight and Frost 1991, Knight 1992, Guiral and Rougier 2007). For the first time, this study also reports the predation of the fourth-instar mosquito larvae by U. aurea, although at lower rates, which could be attributed to their lower mobility, especially in the late fourth-instar stage with ceased feeding before pupation (reduced interaction with Utricularia) and bigger size.

The major developmental habitats of Ae. aegypti and An. stephensi are water-containing cisterns, wells, overhead and underground tanks, curing pits at construction sites, gutters in the roof, fountains, drums, and ornamental tanks, which are rarely cleaned (Thomas et al. 2016). Utricularia aurea exhibited tremendous potential in controlling the larvae of Ae. aegypti and An. stephensi, in curing water pits at construction sites. The predatory potential of U. aurea in the field depends on the ratio between the number of traps and the density of mosquito larvae. Future studies should evaluate the utility of Utricularia in the settings mentioned above in addition to curing water pits.

The advantages of using Utricularia as a biolarval control agent are detailed below. Utricularia has been shown to selectively target mosquito species and not affect nontarget organisms in man-made water storage containers (Couret et al. 2020). Furthermore, water bodies with Utricularia are preferred oviposition sites for odonates, the predators of mosquitoes (Sawchyn and Gillott 1975). Utricularia is highly resistant to insecticides, herbicides, and pesticides (Hanson 1952, Smith and Pullman 1997, Brewer 1999), therefore, can be used in combination with other chemical control agents (Couret et al. 2020). The distribution of diverse Utricularia species across different geographical regions (Gordeev and Sibataev 1995, Ogwal-okeng et al. 2013, Poppinga et al. 2015) negates the need to introduce new non-native species for larval control (Couret et al. 2020).

Even though Utricularia holds a lot of promise in the control of mosquito larvae in both natural and man-made breeding habitats, robust ecological studies on U. aurea must be carried out to evaluate its invasiveness in natural waters, seasonal distribution, and effect on the food chain. In the present study, we observed U. aurea traps to be higher and well developed in December, January, and February. However, the peak malaria season in southwest India is from June to September, and the peak disease transmission season may not coincide with the predatory period of the U. aurea. To effectively control the transmission of mosquito-borne diseases using U. aurea as a biological agent, the plants must grow abundantly in the waters during the transmission seasons of malaria and dengue. Overall, U. area holds a lot of promise as a biolarval agent, and future studies should explore its efficacy in different natural settings.

Conclusion

Utricularia aurea L, a widely distributed aquatic carnivorous plant in India, exhibits predatory activity in the larval stages of the mosquito vectors of public health importance. Therefore, U. area is a promising biolarval control agent, and future studies should explore its predatory efficacy in different natural settings.

Acknowledgments

The authors are thankful to Mr. Prathamesh, Mr. Shamu, Mr. Jagadishwaran, Mr. Bhalchandra, and Mr. Pritesh for their help in the collection of plant material and maintaining the larval population. We are thankful to ICMR-National Institute of Malaria Research for the research facilities.

Author Contributions

Ajeet Mohanty (Conceptualization [Equal], Data curation [Equal], Formal analysis [Equal], Investigation [Equal], Supervision [Equal], Writing—review & editing [Equal]), Abhishek Govekar (Methodology [Equal], Writing—original draft [Equal]), Raja Vukanti (Data curation [Equal], Formal analysis [Equal], Writing—review & editing [Equal]), Justin Montemarano (Data curation [Equal], Formal analysis [Equal], Software [Equal], Writing—review & editing [Equal]), Charles de Souza (Methodology [Equal], Writing—original draft [Equal]), Abhipsa Mohapatra (Conceptualization [Equal], Data curation [Equal], Formal analysis [Equal], Writing—review & editing [Equal]), Janarthanam Kuppuswamy (Conceptualization [Equal], Data curation [Equal], Formal analysis [Equal], Writing—review & editing [Equal]), and Praveen Balabaskaran Nina (Conceptualization [Equal], Data curation [Equal], Formal analysis [Equal], Writing—review & editing [Equal])

References