Physical and chemical cues allow mosquitoes to assess the suitability of potential larval habitats and hence influence the acceptance of oviposition sites (Blackwell and Johnson 2000, McCall 2002, Abrell et al. 2005, Herrera-Varela et al. 2014). Physical cues originate from vegetation, moisture, optical density, color/contrast, temperature, and texture of the substrate (McCrae 1984; Savage et al. 1990; Clements 1999; Koenraadt et al. 2003; Huang et al. 2006b, 2007; Reiskind and Zarrabi 2012). Chemical cues are produced by microorganisms (Trexler et al. 2003, Huang et al. 2006a, Lindh et al. 2008), conspecific eggs (Laurence and Pickett 1982, 1985; Ganesan et al. 2006), conspecific larvae (Mendki et al. 2000, Mokany and Shine 2002, Seenivasagan et al. 2009), water conditioned by eggs and/or larvae (Zahiri et al. 1997, Allan and Kline 1998, Zahiri and Rau 1998), plant infusions (Olagbemiro et al. 2004; Burkett-Cadena and Mullen 2007; Ponnusamy et al. 2010a, b; Tennyson et al. 2012), odors from pollen (Wondwosen et al. 2017), and predators (Mokany and Shine 2002, 2003; Silberbush et al. 2015; Why et al. 2016).

Gravid mosquitoes may be attracted to or deterred by habitats containing conspecific larvae, because the presence of such larvae may indicate the suitability of habitats, an important factor in maximizing the fitness of their offspring (Blaustein and Kotler 1993; Allan and Kline 1998). Studies on the influence of conspecific eggs/ larvae on site selection suggest that culicines are generally attracted to conspecifics (Laurence and Pickett 1982, 1985; Mendki et al. 2000). So far, studies on anophelines have given conflicting results regarding the influence of conspecifics on oviposition behavior. Some authors have reported oviposition deterrence in the presence of conspecific larvae (Bentley and Day 1989; Munga et al. 2006), whereas others have reported that several females may oviposit at the same site (Chen et al. 2006, 2008), which suggests attraction. Olfactory mediation of oviposition behavior in Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) by conspecific larva was reported, and a density-dependent factor has been suggested to regulate this behavior (Sumba et al. 2008). In these studies, no difference was made between the effects of larval stages which may determine the decision taken by the ovipositing female.

The co-existence of various developmental stages of mosquito larvae (instars) in a breeding site may lead to resource competition and cannibalism (Koenraadt and Takken 2003). First instars of the malaria vector An. gambiae s.s. have been reported to be consumed by fourth instars of the same species. It is therefore plausible for a gravid mosquito to avoid oviposition in sites where late instars are present. Therefore, mosquitoes must make a careful assessment of breeding sites before selecting one for their offspring. It has been shown, however, that mosquitoes tend to avoid oviposition in habitats with predators and competitors (Kiflawi et al. 2003, Mokany and Shine 2003). Therefore, mosquitoes are faced with the challenge of choosing a suitable habitat while minimizing the costs of intra- and/or interspecific competition (Blaustein et al. 2004). However, it is not clear whether gravid females can express an optimal oviposition strategy by using only olfactory cues or in combination with other cues emitted from the larval habitat.

The ability of a female mosquito to assess breeding site suitability relies on her ability to detect and estimate the presence and density of conspecifics, in a single breeding site as well as within and among surrounding potential sites. The contribution of breeding sites with high or low larval density to the adult population and to the epidemiology of vector-borne diseases is vital as density-dependent effects may mediate the fitness of mosquitoes emerging from the site. The mechanism, with which a gravid female recognizes various densities of larvae in the breeding site, is not well understood. Only few studies have investigated oviposition site-selection behavior, despite its large consequences for individual fitness, population dynamics, and community structure (Blaustein et al. 2004).

In insects, infochemicals can originate from specialized secretory glands, body orifices, organs involved in digestion and reproduction (e.g., mouth, anus, aedeagus) (Wertheim et al. 2005). Also infochemicals can be emitted by microorganisms living symbiotically with larvae, and attract or repel conspecific adults. Odors from bacteria are known to mediate oviposition behavior in mosquitoes (Ponnusamy et al. 2008, 2010a).

Studies with the mosquito Culiseta longiareolata Macquart (Diptera: Culicidae) (Silberbush et al. 2010) and An. gambiae (Warburg et al. 2011) reveal that gravid females avoid larval habitats containing the cues of the predatory backswimmer, Notonecta maculata Fabricus (Hemiptera: Notonectidae), as a result of detecting the hydrocarbons (n-heneicosane and n-tricosane) produced by N. maculata. On the other hand, an oviposition pheromone n-heneicosane (C21) was identified and characterized from the larval cuticle of Aedes aegypti (L.) (Diptera: Culicidae) (Mendki et al. 2000). Furthermore, it was confirmed that low doses of n-heinecosane attract gravid Ae. aegypti, whereas at higher doses they are repelled by the pheromone (Seenivasagan et al. 2012). It is not surprising that this same compound repels Culiseta. Recent studies based on electroantennographic responses (GC-EAD) and additional oviposition assays confirmed n-heneicosane to be an oviposition pheromone in Ae. aegypti and a behavior modifier of Ae. albopictus (Skuse) (Diptera: Culicidae) in larval habitats (Gonzalez et al. 2014).

Understanding mosquito oviposition behavior can provide a tool for behavioral manipulation of mosquitoes in the field and enable the development of an effective vector surveillance and control strategy. The objective of the present study was to investigate the influence of larval stages and density of conspecifics on the oviposition strategy of An. coluzzii Coetzee & Wilkerson (Diptera: Culicidae) and whether this strategy was chemically mediated. Specifically, the study aimed to explore the age at which larvae attract or deter conspecific gravid females to oviposit, and identify respective intraspecific cues involved in oviposition site selection.

Materials and Methods

No-Choice Experiment

The aim of this experiment was to assess the response of ovipositing females to larvae of different instars (L1 and L4) in the absence of alternative oviposition sites. Individual gravid mosquitoes were exposed to a cup containing either 100 first instars or 50 fourth instars for 72 h. Oviposition was checked at 12, 24, 48, and 72 h. Any eggs remaining in the ovaries and not oviposited were counted following dissection.

Fig. 1.

Diagram of the setup of the double cup experiment in which visual cues from larvae are hidden from the mosquito view by filter paper.

A flow diagram of the experimental procedures is presented in  Supp Fig. 1 (online only).

Results

Oviposition Response to LCW

There was no significant difference in the number of eggs that females deposited in the control cup and the cup with water conditioned by first instars (N = 17, z = –0.24, P = 0.981) or by fourth instars (N = 17, z = –0.118, P = 0.906) (Fig. 3). Also, in a dual-choice test, there was no significant difference between eggs laid on water conditioned by first or fourth instars (N = 17, z = –0.379, P = 0.705) (Fig. 3).

Fig. 2.

(A) Number of eggs laid by single female Anopheles coluzzii per cup in a dual-choice essay, using cups with conspecific first instars in ascending densities and cups containing water only (controls). Median and quartiles are given; asterisks indicate statistical differences between treatment and control for a given density (*P < 0.05, **P < 0.001, Wilcoxon signed rank tests). The number of replicates was 17. (B) Number of eggs laid by single female An. coluzzii per cup in a dual-choice essay, using cups with conspecific fourth instars in densities of 10 and 50 per cup, respectively, against cups containing water only (controls). Median and quartiles are given; asterisks indicate statistical differences between treatment and control for a given density (*P < 0.01, **P < 0.001, Wilcoxon signed rank tests). The number of replicates was 17. (C) The number of eggs laid by An. coluzzii in a dual-choice test between cups treated with low and high densities of conspecific first and fourth instars. Median and quartiles are given; asterisks indicate statistical differences between the larval stages at a given density (*P < 0.001, Wilcoxon signed rank tests). The number of replicates was 17.

Oviposition Response in Single Cup (No-Choice Experiment)

In 15 replicates, 100% of females exposed to cups with first instars laid eggs. By contrast, of the females exposed to fourth instars, only 73.3% laid eggs. Cups with first instars received significantly more eggs 103.4 ± 10.7 (Kruskal–Wallis test, P < 0.001) than cups with fourth instars, which received an average number of 7.6 ± 3.5 eggs. Moreover, all eggs in cups containing first instars were laid within the first 12 h after exposing gravid mosquitoes to the developing larvae, while females exposed to fourth instars spread their eggs between 12 and 48 h. After 72 h, all mosquitoes were removed and dissected. The mean number of eggs retained in mosquitoes exposed to fourth instars (77.5 ± 12.3) was significantly greater (P < 0.001) than the mean number of eggs retained in mosquitoes exposed to first instars (0.5 ± 0.35) (Fig. 5).

Fig. 3.

Anopheles coluzzii oviposition (measured as the number of eggs laid per female) in cups treated with LCW and respective controls. Set 1 consists of LCW from 50 first instars and a control, set 2 consists of LCW from 50 fourth instars and a control, set 3 consists of LCW from 100 fourth instars and a control, and set 4 consists of LCW from 50 first instar larvae and LCW from 50 fourth instar larvae. Median and quartiles are given; there were no significant differences in any of the four treatments (Wilcoxon signed rank tests). The number of replicates was 20.

Table 1.

Oviposition response of Anopheles coluzzii in tcw and larval instars to which tcw has been added

Discussion

Our study indicated that gravid females of An. coluzzii were affected in their oviposition behavior by volatiles produced by conspecific larvae, in which first instars produced attractive chemicals, whereas fourth instars produced chemicals that deterred females. This is likely the reason why females select sites with a high density of first instars, while avoiding sites with low densities of such larvae. Studies on An. gambiae oviposition behavior showed that members of this group of anophelines tend to lay eggs in groups and several females may oviposit in the same site (Chen et al. 2006, 2008). Therefore, the choice for laying eggs in sites with high densities of conspecifics can be beneficial, unless these sites confer unfavorable traits such as competition, cannibalism, or predation. When these conspecifics, however, are in an advanced age of development, they may be dangerous for young siblings, and females avoid such sites to reduce the risk of cannibalism (Koenraadt and Takken 2003). Therefore, the avoidance of oviposition in sites where late-stage larvae are present is of great advantage to mosquitoes and reveals how mosquitoes might achieve an optimal oviposition strategy by exploiting sites that confer the best chance for offspring development and survival. In nature, the presence of immature stages could indicate the suitability of that site in terms of food, site persistence, lack of predation, and appropriate abiotic conditions (Wong et al. 2011).

The findings of our experiment suggest that in the absence of conspecific larvae, mosquitoes oviposit in water bodies selected at random. The mean number of eggs laid in cups with water only was less than the mean number of eggs laid in cups containing larvae with densities starting from 1.7 larvae per ml of water. This indicates that females retain some of their eggs when there is no clear evidence of site suitability. Moreover, in the presence of first instars, oviposition responses increased with increasing larval density starting from 1.7 larvae per ml of water. Likewise, the strength of deterrence caused by fourth instars increased with increasing larval density. We can therefore conclude that the oviposition response in the presence of conspecific larvae is density-dependent. This was also found by Ogbunugafor and Sumba (2008), who reported similar olfactionguided oviposition behavior of An. gambiae with density dependence. In Ogbunugafor and Sumba's study, however, no distinction was made between young and older instars. In fact, their study reported deterrence of oviposition by all larval densities in distilled water. As we learned from our study, this is possibly caused by volatiles produced by fourth instars, which under all circumstances, at densities of 0.3–3.3 larvae per ml, caused deterrence. Our findings also supported an earlier hypothesis that breeding sites with low larval density are perceived as unsuitable by gravid mosquitoes (Munga et al. 2006).

Fig. 4.

Anopheles coluzzii oviposition (measured as the number of eggs laid per female) test between cups treated with 1) 100 first instars and a control, 2) 100 fourth instars and a control, and 3) between 100 first instars and 100 fourth instars using a double cup setup. Median and quartiles are given (*P < 0.05, Wilcoxon signed rank tests). The number of replicates was 17.

Higher larval densities are common, especially in small breeding sites such as hoof/foot prints, tire prints, and road banks. In a nutrient-enriched site, a single mosquito may lay 50–500 eggs at one time (Clements 1992). As conspecific sharing of breeding sites is common among mosquitoes (Chen et al. 2008) and egg-hatching rate is high (Phasomkusolsil et al. 2013), small sites are often harboring high larval densities. Therefore, the existence of breeding sites with low larval density suggests unfavorable conditions. Anopheles gambiae s.l. tends to breed in such small, shallow, and temporary sites due to absence of predation (Service 1993, Mala and Irungu 2011, Muriu et al. 2013).

In natural conditions some habitats contain high densities of anopheline larvae, whereas many others have none despite high densities of adult mosquitoes in the immediate environment (Minakawa et al. 2002). These observations are in line with the findings from our study and observations by other authors that some aquatic habitats are more attractive or suitable for oviposition and larval development than others (Minakawa et al. 1999), and as a result, mosquitoes perform selective oviposition behavior (Davis et al. 2015). Randomly amplified polymorphic DNA used to estimate the number of full-sibling family size revealed that the average family size of Ae. aegypti mosquito larvae in a container is 11 and the family size distribution among containers is skewed toward containers with one or two families (Apostol et al. 1994). Studies on oviposition behavior of An. gambiae using a pairwise genetic relatedness method substantiate that average genetic relatedness tends to be low for breeding sites with high larval populations (Chen et al. 2006). This means that An. gambiae prefers to oviposit where others have oviposited. Our study suggests that the presence of an optimum density of first instar stage larvae in selected breeding sites may induce other conspecific mosquitoes to oviposit in the same breeding sites.

Results from the no-choice experiments suggest that substrates containing first instars attract egg-laying behavior of conspecific gravid females, whereas substrates containing fourth instars deter females intending to oviposit and induce egg-retention behavior. Egg retention is common in skip oviposition where mosquitoes do not lay all eggs in one site but retain some (Williams et al. 2008) and lay in multiple sites (Colton et al. 2003, Reiter 2007, Snell et al. 2010).

In our study we found that egg retention is common when fourth instars are present in oviposition cups, and oviposition in more than one container occurred when nutrient-poor substrates were used in both oviposition choices. This suggests that skip oviposition occurs when there is no clear indication of site suitability, as mosquitoes do not display a clear preference. Moreover, our results suggest that in natural settings, where the only available water body contains predators (e.g., conspecific fourth instars), An. coluzzii will lay few eggs in that risky environment on the first day. On the second day, An. coluzzii will lay even fewer eggs as compared to the first day while retaining the rest of the eggs. These observations are akin to skip oviposition behavior, which is displayed by other mosquitoes. For example, in Ae. aegypti, significantly higher oviposition occurred in one site and residual eggs were distributed in groups of 1–30 eggs (Oliva et al. 2014).

Fig. 5.

Egg-laying responses of Anopheles coluzzii when exposed to oviposition substrates containing either first instar larvae (L1) or fourth instar larvae (L4) under no-choice conditions (*P < 0.001). The number of replicates was 15.

Our data from the double cup experiment, where visual stimuli from water and/or larvae were excluded, indicate that olfactory compounds are involved in oviposition behavior. Chemical communication has been demonstrated to operate in mosquito oviposition behavior in several mosquito species (Bentley and Day 1989, McCall and Cameron 1995) including An. gambiae s.s. (Blackwell and Johnson 2000), a sibling of An. coluzzii. However, our results demonstrate that only alive, conspecific larvae produce these cues, as LCW did not cause a behavioral response. These effects are clearly dose-dependent, as the magnitude of the influence increases with increasing larval density and at low larval densities the influence was not observed. Ogbunugafor and Sumba (2008) also reported the presence of oviposition-mediating olfactory cues derived from immature larvae of An. gambiae. In their study, however, larval water from different instars was mixed, and hence the chemical cues from one instar may have overridden the deterrent effects from fourth instars.

The effects of the larval cues appeared to be overruled by cues produced by Tetramin. Nutritional substrates are well known to affect An. gambiae oviposition behavior (Lindh et al. 2008) and an oviposition attractant cedrol was identified and associated with the presence of microbes in the breeding habitat (Lindh et al. 2015). Therefore, it is likely that chemical cues produced from nutritional constituents such as Tetramin impact mosquito oviposition behavior, masking the effects of the larval cues. Significant differences in cues from first and fourth instars, however, are evident as gravid females laid more eggs in larval substrates with first instars than with fourth instars.

When female An. coluzzii were offered a choice between oviposition cups with fourth instars and a control, they deposited more eggs in the control cups. When subjected to a choice between same high densities of early- and late-stage larvae, female mosquitoes opted for the former exclusively. These results indicate the presence of different types of larval pheromones involved in the oviposition strategy of An. coluzzii. Pheromones produced by early instars that signal to the gravid female suitability of the site and pheromones produced by late instars that signal unsuitability. The pheromone might be of the same nature, but produced at a different concentration by early- and late-stage larvae. On the other hand, the difference between eggs laid in cups with low densities of first and fourth larval instars was not significant, and confirms our earlier findings that a low density of first instars does not signal the quality of a site. It is possible that the emission of chemical cues that are likely to mediate oviposition behavior is too low when larval density is low.

Under laboratory conditions, gravid An. gambiae usually touch the oviposition substrate briefly or hover 5–10 cm above the site before depositing her eggs (Huang et al. 2006b). Such an assessment procedure may involve visual, tactile, or olfactory cues, alone or in combination. Experiments with LCW did not show a significant difference between conditioned water and controls. The disappearance of attractiveness or deterrence after the removal of larvae may suggest two things: first, vision is also involved in the previously displayed behavior. Secondly, the presence of live larvae is necessary to evoke the observed behavior. Chemicals released by mosquito larvae are highly volatile and present in low concentrations (Allan and Kline 1998). Therefore, it is possible that the removal of larvae and volatilization of chemicals have contributed to absence of attractiveness or deterrence response in the LCW experiments.

Studies by Huang et al. (2018) emphasized that cannibalization of newly hatched An. gambiae larvae by fourth instar larvae was a result of egg reduction in egg counts and not olfactory deterrent cues. However, results from the double cup experiments, in which the larvae are hidden from view by a filter paper (Fig. 1), suggest that chemical cues are involved in the oviposition behavior of An. coluzzii. Our study further suggests that oviposition-deterrent chemicals are responsible for counteracting resource competition and cannibalism among larvae of An. coluzzii in field settings. We therefore confirm recent findings that ovipositing An. coluzzii females are less inclined to lay eggs in pools that have late instars compared to those with early instars (Sumba et al. 2008). These observations can partly explain why an initial population peak of Anopheles larvae observed at a fixed point in a rice field was not immediately followed by a sustained high population peak of early instars (Mutero et al. 2004). Our study was conducted in the laboratory, using mosquitoes that originated from Liberia. When the study was repeated in Tanzania using mosquitoes that originated from Tanzania, we obtained similar results (V. S. Mwingira, unpublished data). However, further studies need to be conducted in various places with different climatic and geographic conditions to clarify the existence of a region-specific cue in An. coluzzii (Ogbunugafor and Sumba 2008).

Larval-based cues can be used for manipulation of mosquito behavior in a push-pull system by making protected resources unsuitable to mosquitoes (by repellents) while luring them toward attractive sources (by attractants) where they can be eliminated by insecticides. It appears evident that larval hydrocarbons can be detected by conspecific (Seenivasagan et al. 2009) and heterospecific mosquitoes (Gonzalez et al. 2014). Moreover, depending on the concentration, female mosquitoes were attracted to a low dose, while higher doses enforced repellency (Seenivasagan et al. 2009). We suspect that the observed pattern in our study might be explained by a similar mechanism whereby early instars emit chemical cues at low concentrations that attract, while late instars emit high concentrations which repel female mosquitoes.

Supplementary Data

Supplementary data are available at Journal of Medical Entomology online.