Rapid habituation by mosquito larvae to ... - Wiley Online Library

Vol. 39, no. 2

Journal of Vector Ecology

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Rapid habituation by mosquito larvae to predator kairomones Derek Roberts Biology Department, Sultan Qaboos University, PO Box 36, Al-Khod 123, Oman, [email protected] Received 1 July 2014; Accepted 22 July 2014 ABSTRACT: Larvae of some species of mosquitoes have been shown to respond to water-borne kairomones from predators by reducing bottom-feeding and replacing it with surface filter-feeding, which uses less movement and is thus less likely to attract a predator. However, if no predator attack takes place, then it would be more efficient to use a risk allocation strategy of habituating their response depending on the predator and the overall risk. The larvae of Culiseta longiareolata Macquart live in temporary rain-filled pools, where they are exposed to a high level of predation. Within one hour, they responded to kairomones from dragonfly or damselfly nymphs, or to the fish Aphanius, by significantly reducing bottom-feeding activity. Continued exposure to the predator kairomones resulted in habituation of their response to damselflies, a slower habituation to fish, but no habituation to dragonflies even after 30 h. In contrast, the larvae of Culex quinquefasciatus Say normally live in highly polluted and thus anaerobic water, where the predation risk will be much lower. They also showed a significant reduction in bottomfeeding after 1 h of exposure to predator kairomones but had completely habituated this response within 6 h of continuous exposure. Some species of mosquito larvae can thus show a very rapid habituation to predator kairomones, while others only habituate slowly depending on the predator and overall predation risk. Journal of Vector Ecology 39 (2): 355-360. 2014. Keyword Index: Culiseta longiareolata, Culex quinquefasciatus, predator kairomones, habituation, filter feeding, mosquito larvae.

INTRODUCTION Extensive studies have shown that aquatic prey have specific defenses against predators and that these defenses can be adapted depending on the predator risk (see review by Ferrari et al. 2010). Since predator defenses usually have a metabolic cost, being able to habituate a behavioral response, when the predator risk is low, will be advantageous to the prey (Ydenberg and Dill 1986). In an aquatic environment, many prey organisms rely on detecting the presence of predators through their waterborne kairomones, since vision among vegetation and through disturbed water may be limited (Dodds and Whiles 2010). A common response to predator kairomones is for the prey to reduce its activity, so that it is less visible to the predator. Mosquito larvae have been shown to respond in this way to the presence of dragonfly nymphs (Roberts 2012), notonectids (Knight et al. 2004, Beketov and Liess 2007), and fish (Bond et al. 2005). However, reducing their activity will make feeding less efficient, so this trait compensation can slow down growth and produce adults that are smaller, and so less competitive (Roberts 2012). Other aquatic insects have been shown to use different anti-predator strategies. Thus Odonata may avoid the location of predators (Pierce 1988) or increase nocturnal feeding (Koperski 1997), when there is less chance of being detected. Further predator-mediated indirect effects shown in aquatic organisms include morphological changes in Daphnia (Barry 1994) and in tadpoles (Relyea 2001) to make the prey less easy to eat. These behavioral and morphological changes require metabolic costs that result from physiological changes in the way energy is stored, in the production of stress proteins, and antioxidant enzymes that allow a prey to respond more rapidly to a predator attack

(Stoks et al. 2005, Slos and Stoks 2008). Because of the physiological costs of these predator defenses, habituation, a behavioral plasticity in which the prey learns not to respond to low risks of predator attack, should give an energetic advantage. Habituation to predators among vertebrates is well studied and recent research includes: kangaroo habituation to unnatural sounds (Biedenweg et al. 2011), seals habituating to populations of killer whales that do not feed on seals (Deecke et al. 2002), blackbirds habituating to humans that do not attack (Rodriguez-Prieto et al. 2009), and lizards habituating to artificial attacks (Rodriguez-Prieto et al. 2011). There have been a number of studies on insect habituation of predator behavioral defenses. Some examples are: bats use echolocation to become major predators of nocturnal moths and as a result, moths have evolved hearing organs with which they have a range of behavioral strategies to counter bat attacks (Yager 2012), but continuous exposure to ultrasound results in moths habituating and ignoring the sound (Gillam et al. 2011). Similarly, crickets (which also have hearing organs) habituate to continuous bat ultrasounds (Engel and Hoy 1999). Colonies of aphids respond to attack by predatory Coccinelid beetles by producing an alarm pheromone that triggers a range of defensive responses in the aphids, but in continuous presence of this alarm pheromone they failed to respond (Vos et al. 2010). Davis and Heslop (2004) showed that pet hissing cockroaches were able to habituate their alarm response to specific humans. However, there has been little research on habituation in mosquitoes. Fish, in particular, have been used in the biological control of certain species of mosquito larvae (Bence 1988, Chandra et al. 2008). The fish Aphanius was also an important component in Oman’s eradication of malaria by controlling the vectors Anopheles


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culicifacies Giles and An. stephensi Liston (pers. comm.) Mosquito species with a more efficient response to predatory fish may reduce the effectiveness of this control method. In this study, two mosquito species were compared. Culex quinquefasciatus Say is a specialized species largely restricted to urban areas in Oman (Roberts and Irving-Bell 1997), where it mainly breeds in septic tanks (Menon and Rajagopalan 1980). Culiseta longiareolata Macquart is found in temporary rain-filled pools (Spencer et al. 2002). Culex quinquefasciatus thus has little exposure to predators by living in very anaerobic water where predators would find it difficult to survive, while Culiseta longiareolata is faced with a high level of predation by a range of species, both fish and different insects. These two mosquito species had previously been shown (Roberts 2014) to respond differently to predator kairomones, with Culiseta longiareolata reducing the more risky active bottom-feeding for the safer more inactive surface filter-feeding in the presence of predator kairomones. However, those experiments were carried out after about 20 h of exposure to the kairomones. The present experiments thus investigated whether this response could have been affected by a rapid habituation within this 20 h period, and if so, whether this habituation varied between the two mosquito species and depended on the predator. MATERIALS AND METHODS Source of mosquitoes and predators Culex quinquefasciatus egg rafts were collected from the Sultan Qaboos University Botanic Garden (Oman) every day and kept separately in the laboratory at 24° C until they hatched. Culiseta longiareolata egg rafts were collected weekly from rain-filled rock pools in Wadi Qurai at Sumail in the Jebel Akhdar mountains, 60 km from the university campus. Dragonfly nymphs (Crocothemis erythraea Brullé) and damselfly nymphs (Ischnura evansi Morton) were collected from very small (< 2m) fish-free pools in Wadi Al-Khod, about 5 km from the university. Aphanius dispar Ruppel fish were also collected from Wadi Al-Khod. During the experiments, only final instars of the dragonfly and damselfly nymphs were used. The Aphanius were mature females. Between experiments, the predators were fed on final (4th) instars of the appropriate mosquito species. Cu. longiareolata were studied first (May, 2013), then Cx. quinquefasciatus were studied in June, 2013. Experimental design Both mosquito species were tested at a laboratory temperature of 23° C. In each replicate, four sets of 30 4th instar mosquito larvae were exposed to, respectively, i) conditioned tap water (negative control); ii) water from Aphanius fish; iii) water from damselfly nymphs; iv) water from dragonfly nymphs. However, when the Cx. quinquefasciatus were being tested, insufficient dragonfly larvae could be collected, so they were not tested against this mosquito. There were ten replicates. The predators were each kept in 4 liters of conditioned tap water in a 16 cm diameter plastic container. Water from

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each predator bottle was pumped using Welco® peristaltic pumps (WPX1 from Welco Ltd, 331 Sumiyoshi-cho, Tokyo, Japan) that had been calibrated to a flow rate of 70 ml/h into one of the mosquito jars. Each jar of 30 mosquito larvae was a polystyrene 8 cm diameter jar containing 400 ml water that overflowed into a bucket. The overflow spout was covered with insect netting to prevent escape of the mosquito larvae. After use, the predators were removed to holding containers, so that different individual predators were used in each replicate. The predators were each put in to their 4 one-predator bottles on the afternoon before the experiment, to give time for predator kairomones to accumulate, and each were immediately fed with four mosquito larvae. The predators remained in their bottles until the end of the replicate. At the same time, 30 4th instar mosquito larvae were put in to each mosquito jar, giving a ratio of 0.075 larvae per ml of water, which is well below levels of crowding that might affect the mosquito (Roberts and Kokkinn 2010). The larvae were then fed with yeast powder at a dosage of 0.09 mg/larva. The pumps were started at 09:00 the next morning and each of the predators was immediately fed with another four mosquito larvae (at -1 h) and later fed again on the 2nd day (at 24 h in to the experiment). No readings were initially taken, to allow time for the predator chemicals to disperse through the mosquito larval jar and to allow the larvae to become habituated to the disturbance of the water dripping in and draining out of their container. i) Initial readings started at 10:00 (one h after the pumps started). Fifteen readings were taken at 5-min intervals for each of the mosquito containers. The number of mosquito larvae filter feeding at the surface were counted and compared with the number of larvae scraping biofilms on the sides and bottom. Replicates were repeated on subsequent days with fresh sets of mosquito larvae and of predators, to give a total of ten replicates (each with 15 readings). ii) A further 15 readings of the same mosquito larvae were taken at 15:00 (6 h of continuous exposure to the chemicals). iii) Final readings were taken the next day at 15:00 (30 h of chemical exposure) for the same mosquito larvae. Data analysis The data were given arcsine transformations. Analysis was by a general linear model ANOVA with Tukey’s post-hoc analysis using SPSS software (SPSS for Windows 10.0.0, 1999) for repeated measures. The data were then back-transformed to percentages for plotting the graphs. RESULTS Over the 30-h period of the experiment, the control larvae showed no significant change in their behavior (F = 0.18; df = 2, 26; p = 0.84) for either the initial experiment with Cu. longiareolata (Figure 2), or the subsequent experiment with Cx. quinquefasciatus (Figure 4). There were thus no circadian laboratory effects biasing the experiments. After the Cu. longiareolata larvae had been exposed to the pumped chemicals for 1 h (Figure 1), bottom-feeding

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Figure 1. Culiseta longiareolata: Effect of exposure to either damselfly or Aphanius fish kairomones on the mean (± SE) % of 4th instar larvae that are bottom feeding in comparison with controls, after 1 h, 6 h, and 30 h exposure. Means with the same letter are not significantly different (Tukey’s HSD test; p < 0.05).

Figure 2. Culiseta longiareolata: Changes in mean (± SE) % of 4th instar larvae that are bottom feeding after 1 h, 6 h, and 30 h, when they are controls (exposed to clean water), exposed to chemicals from damselfly nymphs, or exposed to chemicals from the fish Aphanius. Means with the same letter are not significantly different (Tukey’s HSD test; p < 0.05). activity by larvae exposed to either damselfly, dragonfly, or fish kairomones was significantly reduced (8.30 ± 1.00, 6.00 ± 0.85, 5.20 ± 1.14%, respectively) compared with the 14.62 ± 2.73% of the control larvae (F = 9.10; df = 3, 34; p = 0.0001). However, the response to damselflies was less than to the other two predators (F = 5.03; df = 1, 16; p = 0.04). After 6 h of exposure, bottom-feeding had increased sufficiently that only the dragonfly nymphs were still significantly lower than the controls (F= 9.47; df = 1, 16; p = 0.007). By 30 h of exposure, there was a further increase in bottom-feeding, but bottom-feeding by those exposed to dragonflies remained significantly lower than the controls (F = 6.10; df = 1, 16; p = 0.025). After the Cx. quinquefasciatus larvae had been exposed to the pumped chemicals for 1 h (Figure 3), bottom-feeding activity by larvae exposed to either damselfly or fish kairomones was significantly reduced to 25.25 ± 1.96%, compared with

the 35.36 ± 4.30% of the control larvae (F = 5.47; df = 2, 26; p = 0.01). However after 6 h (Figure 4), larvae exposed to either damselfly or fish kairomones had increased bottom-feeding to 30.30 ± 2.54 % and 32.70 ± 5.22%, respectively, and were thus no longer significantly different from the controls (F = 0.65; df = 2, 26; p = 0.53). They remained not significantly different (F = 0.23; df = 2, 26; p = 0.80) from the controls at 30 h. Figure 4 shows the significant rise in bottom-feeding activity from 1 h to 30 h for both the mosquito larvae exposed to damselfly kairomones (in the repeated measures ANOVA, F = 11.82; df = 2, 26; p = 0.004) and those exposed to fish kairomones (F = 4.26; df = 2, 26; p = 0.03). However, although the mosquito larvae showed a habituation to both predators, the response was much greater to the damselfly larvae. Culiseta longiareolata larvae thus showed only a slow and small habituation within the 30-h time period for exposure to fish and damselflies but no significant habituation to

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Figure 3. Culex quinquefasciatus: Effect of exposure to either damselfly or Aphanius fish kairomones on the mean (± SE) % of 4th instar larvae that are bottom feeding in comparison with controls, after 1 h, 6 h, and 30 h exposure. Means with the same letter are not significantly different (Tukey’s HSD test; p < 0.05).

Figure 4. Culex quinquefasciatus Changes in mean (± SE) % of 4th instar larvae that are bottom feeding after 1 h, 6 h, and 30 h, when they are controls (exposed to clean water), exposed to chemicals from damselfly nymphs, or exposed to chemicals from the fish Aphanius. Means with the same letter are not significantly different (Tukey’s HSD test; p < 0.05).


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dragonflies. The Cx. quinquefasciatus larvae had a very rapid habituation to both predators. DISCUSSION Being able to detect the presence of a predator by its kairomones allows a mosquito larva to reduce the risk of predation by altering its activity (Roberts 2012). This phenotypic plasticity comes at a cost of reduced feeding efficiency and increased metabolic cost (Stoks et al. 2005), which will affect its development. As a result, not all mosquito species respond to the presence of predators, and their response will depend on their perceived predation risk. Thus Sih (1986) showed that the container-breeding Aedes aegypti L. showed only a weak response to Notonecta, because it normally has little exposure to them, while Culex pipiens L. had a strong response. Similarly, Kesavaraju and Juliano (2004) showed that native Aedes triseriatus Say strongly responded to the native predator Toxorhynchites rutilis Theobald, but introduced Anopheles albopictus Skuse showed no response. The response is usually specific to the predator, providing they can differentiate between predator kairomones. This ability has been shown in some mosquito larvae that have a greater reduction in bottom-feeding (Roberts 2014) in the presence of dragonfly nymphs, since dragonflies largely hunt on the bottom (Corbet 1980). Bottom-feeding is less affected by damselfly nymphs, since damselflies sit on emerging grass and so may be at any depth. The mosquito larvae showed no response to final instar Nepid larvae that in the lab refused to eat mosquito larvae. Damselfly nymphs (Chivers et al.1996, Stoks et al. 2003) and dragonfly nymphs (Hopper 2001) have similarly been shown to have different responses to various fish predators. These experiments show that the prey are able to identify different predators and do respond more strongly to higher risk predators. One of the problems of using chemical cues for determining the presence of a predator is that in a large pool, the predator may not be nearby. Thus, a reduction in feeding may not be risk effective, so that if a predator attack does not materialize, the mosquito should habituate its response and revert to more efficient bottom-feeding. Some of these kairomones have been shown to be relatively stable and so may not be a good indicator of immediate risk. Thus, Blaustein et al. (2004) showed that adding Notonecta kairomone to a pool inhibited oviposition by Cu. longiareolata for eight days, while van Buskirk et al. (2014) showed that the efficacy of Aeshna dragonfly kairomone had a half-life of 35 h. Some mosquitoes have been shown to have a threat-sensitive response that is graduated depending on the predator kairomone concentration. Examples are the response of Aedes triseriatus to the predatory mosquito Toxorhynchites rutilis (Kesavaraju et al. 2007) and for three species of Anopheles responding to Notonectid predators (Roux et al. 2014). Although Ferrari et al. (2007) showed that this threat-sensitive response depends on the background risk and so varied between different pools. Lima and Bednekoff (1999) proposed the risk allocation hypothesis in which the prey response to a predator should depend on the risk. Thus, the prey should respond stronger

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to a predator, such as a fish, that is only present for a short period and then swims away. They should respond least to a predator that is always present, such as the relatively inactive damselfly larvae, since feeding cannot be discontinued for too long. Culiseta longiareolata showed the least response to damselflies, but habituated more rapidly to fish than to dragonflies (but maybe the bottom-feeding dragonflies are a greater risk to a bottom-feeding mosquito). Overall, Cu. longiareolata, living in a habitat with a high predation risk, showed a slow rate of habituation. Culex quinquefasciatus lives in a specialized habitat, where few predators can survive due to low oxygen levels. Although they initially responded to both predators tested, they very rapidly habituated their initial response. Even within 6 h, their response to predators was no longer significantly different from the controls. Acknowledgments I thank Sultan Qaboos University for providing research facilities and Dr. Michael Barry for assistance in the field collection of predators. REFERENCES CITED Barry, M.J. 1994. The costs of crest induction for Daphnia carinata. Oecologia 97: 278–288. Beketov, M.A. and M. Liess. 2007. Predation risk perception and food scarcity induce alterations of life-cycle traits of the mosquito Culex pipiens. Ecol. Entomol. 32: 405–410. Bence, J.R. 1988. Indirect effects and biological control of mosquitoes by mosquitofish. J. Appl. Ecol. 25: 505-521. Biedenweg, T.A., M.H. Parsons, P.A. Fleming, and D.T. Blumstein. 2011. Sounds scary? Lack of habituation following the presentation of novel sounds. PLoS ONE 6: e14549. Blaustein, L., M. Kiflawi, A. Eitam, M. Mangel, and J.E. Cohen. 2004. Oviposition habitat selection in response to risk of predation in temporary pools: mode of detection and consistency across experimental venue. Oecologia 138: 300–305. Bond, J.G., J.I. Arredondo-Jiménez, M.H. Rodríguez, H. Quiroz-Martínez, and T. Williams. 2005. Oviposition habitat selection for a predator refuge and food source in a mosquito. Ecol. Entomol. 30: 255–263. van Buskirk, J., A. Krügel, J. Kunz, F. Miss, and A. Stamm. 2014. The rate of degradation of chemical cues indicating predation risk: An experiment and review. Ethology 120: 1-8. Chandra, G., I. Bhattacharjee, S.N. Chatterjee, and A. Ghosh 2008. Mosquito control by larvivorous fish. Indian J. Med. Res. 127: 13-27. Chivers, D.P., B.D. Wisenden, and R.J.F. Smith. 1996. Damselfly larvae learn to recognize predators from chemical cues in the predator’s diet. Anim. Behav. 52: 315–320. Corbet, P.S. 1980. Biology of Odonata. Annu. Rev. Entomol. 25: 189–217.

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