Conspecific and allospecific larval extracts entice mosquitoes to lay eggs and may be used in attract-and-kill control strategy

One of the strategies of integrated vector management is to lure gravid mosquitoes for surveillance purposes or to entice them to lay eggs in water containing toxins that kill the offspring (attract-and-kill or trap-and-kill). Typically, the major challenge of this approach is the development of a lure that stimulates oviposition plus a toxin with no deterrent effect. Bacillus thuringiensis var. israelensis (Bti) satisfies the latter criterion, but lures for these autocidal gravid traps are sorely needed. We observed that gravid Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus laid significantly more eggs in cups with extracts from 4th-stage larvae (4 L) of the same or different species. No activity was found when 4 L were extracted with hexane, diethyl ether, methanol, or butanol, but activity was observed with dimethyl sulfoxide extracts. Larval extracts contained both oviposition stimulant(s)/attractant(s) and deterrent(s), which partitioned in the water and hexane phases, respectively. Lyophilized larval extracts were active after a month, but activity was reduced by keeping the sample at 4 °C. In the tested range of 0.1 to 1 larvae-equivalent per milliliter, oviposition activity increased in a dose-dependent manner. In field experiments, Ae. aegpti laid significantly more eggs in traps loaded with larval extracts plus Bti than in control traps with water plus Bti.

Integrated vector management is a combination of environmentally friendly strategies that can be used to prevent transmission of vector-borne diseases 1 . Throughout the world, vector abatement groups monitor populations of native species and possibly invasive species of mosquitoes as well as circulation of previously reported and possibly new pathogens. Typically, they inspect house-to-house for possible mosquito breeding sites and aspire adult mosquitoes to determine if they carry pathogens. More importantly, they trap adult mosquitoes with CO 2 -baited and gravid traps. The physiological state of the mosquitoes captured in these traps differ. CO 2 is a good lure for blood-seeking females mosquitoes, but the largest majority of captured female mosquitoes never had a previous blood meal. Thus, there could be many false negatives in surveillance and early detection of pathogens. By contrast, the gravid traps capture mostly females that already had a blood meal and, consequently, more likely to be infected with a vector-borne pathogen than the general adult population 2 . In addition to monitoring and surveillance, these gravid traps (=ovitraps) have a potential application in IPM for direct control of mosquito populations. For a direct trap-and-kill control strategy, ovitraps may be transformed into autocidal gravid ovitraps by adding a biological agent (eg, Bacillus thuringiensis var. israelensis, Bti), an insecticide, or even an adhesive strip, in addition to a natural or synthetic lure (reviewed in ref. 2 ).
It has been reported for the last four decades that larval-holding water and larval-rearing water are "attractive" to conspecific Aedes and Culex mosquitoes 3-13 , although it has not been unambiguously determined whether these lures are derived from immature stages of mosquitoes, from bacteria they host, or even from bacteria in the rearing medium. From an evolutionary perspective, the cost-benefit of producing such a signal is intriguing, but from epidemiological and practical viewpoints, it is a weak link worth exploring as a target for vector control. Here, we show that gravid females Ae. aegypti, Ae. albopictus, or Culex quinquefasciatus mosquitoes lay significantly more eggs in oviposition cups loaded with aqueous extracts from conspecific or allospecific  For clarity, data are presented in percentage of oviposition preference, with mean number of eggs or egg rafts presented along with each bar. After arcsine transformation and passing the Shapiro-Wilk normality test, each dataset was compared by using the 2-tailed, paired t test. www.nature.com/scientificreports www.nature.com/scientificreports/ A very hydrophobic compound, n-heneicosane 17 , has been isolated from Ae. aegypti eggs and has been demonstrated to stimulate the antennae 18 of both Ae. aegypti and Ae. albopictus and thus has been suggested to be an oviposition attractant 17,18 . We then tested whether the active ingredients could be extracted with organic solvents. To avoid emulsification when the extracts were mixed with water in oviposition cups, hexane extracts were dried up and reconstituted in dimethyl sulfoxide (DMSO). Indeed, there was no significant difference in the number of eggs laid by Ae. aegypti gravid females in cups loaded with hexane extract vs. control cups (Fig. 4A). By contrast, there was a significant preference for cups loaded with DMSO larval extracts compared with the control (water plus DMSO) (Fig. 4B). Similarly, Cx. quinquefasciatus showed a significant preference for DMSO but not for hexane extracts (Fig. 4C,D). We repeated these experiments and noticed a trend of controls getting more egg rafts than hexane extracts, thus suggesting a possible deterrent effect from hexane extracts. We surmised that a trace of these or other deterrents might be contained in our aqueous extracts. To test this assumption, we performed liquid-liquid extraction of the active material and tested separately the aqueous and organic phases. Of note, a small gel-like intermediate phase was discarded after the aqueous phase was collected and before the start of collecting the hexane phase. There was a clear preference for gravid Cx. quinquefasciatus to lay eggs in the aqueous fraction over the control (Fig. 5A), whereas the organic phase showed a deterrent effect (Fig. 5B). www.nature.com/scientificreports www.nature.com/scientificreports/ We, therefore, concluded that the aqueous extracts contain both oviposition stimulant(s)/attractant(s) and deterrent(s) with the former offsetting the latter. We then extracted Cx. quinquefasciatus L4 larvae with other organic solvents and found similar deterrent effects with diethyl ether, methanol, and butanol (Fig. S3). Furthermore, we surmised that the active ingredient is either water-soluble organic compound(s) or protein(s)/peptide(s) that do not require folding for activity otherwise, activity in DMSO extracts would have been lost 19 .
Next, we investigated whether lyophilization would affect activity. Larval extracts from the yellow fever mosquito were separated into two groups; half of the sample was extracted and then kept at 4 °C for three days, and the other half of the sample was lyophilized and three days later extracted just before bioassays. Responses elicited by the refrigerated and lyophilized samples were significantly higher than the responses observed in their respective controls (Fig. 6A,B). Interestingly, however, when these experiments were performed with a longer storage time (30 days), the refrigerated sample lost activity, whereas activity was retained by the lyophilized sample (Fig. 6C,D). These experiments reinforce what has been observed with direct organic solvent extractions. Specifically, it is highly unlikely that the active ingredients are organic molecules of low or medium molecular weight, which would have evaporated during lyophilization. Moreover, these data show that the active ingredient(s) undergoes degradation at 4 °C as would be expected for a peptide or protein kept in a crude extract, which must contain proteolytic enzymes from the mosquito gut.
It is very common in chemical ecology that some compounds act in a dose-dependent manner, being an attractant at lower doses and a deterrent at higher doses. Because we used a standard concentration of 0.33 L-eq/ ml throughout these studies, we next tested lower and higher doses. The activity from 0.1-1 L-eq/ml increased in a dose-dependent manner (Fig. 7). It is therefore unlikely that the oviposition stimulant(s)/attractant(s) in our aqueous extracts are related to overcrowding factors. If overcrowding factors were extracted from larvae, the extracts would lose activity at higher doses (eg, 1L-eq/ml), which would represent an overcrowding environment. The active lures are likely exudates from larvae (and pupae), but we cannot unambiguously determine whether they are derived from bacteria housed in mosquito gut or by the insect.
Lastly, we explored the potential application of these larval extracts in attraction-and-kill strategies. Specifically, we questioned whether these extracts would be active in the field when combined with a toxic agent. The number of eggs in traps loaded with both larval extract and Bti were significantly higher than in the control traps with water plus Bti (Fig. 8). In conclusion, L4 larval extracts have a potential application in integrated vector management. The logistics of this attract-and-kill strategy might be simplified when the active ingredients are identified and synthetic counterparts are used instead of cumbersome crude extracts. For the time being, however, extracts from lyophilized larvae may be used as lure. Cornel's stock laboratory colony, which in turn started from adult mosquitoes collected in Merced, CA, in the 1950s. The Davis colony has been kept for more than 7 years at 27 ± 1 °C, 75% ± 5 relative humidity, and under a photoperiod of 12:12 h (light:dark). The Recife colony of Cx. quinquefasciatus originated from eggs collected in Peixinhos, a neighborhood of Olinda, metropolitan region of Recife, Pernambuco, Brazil in 2009. The Ae. aegypti and Ae. albopictus colonies started in 1996 and 1998, respectively, from eggs collected in neighborhoods in Recife. All 3 mosquito colonies from Brazil were kept in Recife at 26 ± 2 °C, 65-85% relative humidity, and under a photoperiod of 12:12 h (light:dark). Larvae were kept in plastic containers (30 × 15 cm; 10 cm height) with a density of approximately 0.3 larvae/ml. Extraction procedures. Fourth-stage larvae were collected with a plastic mesh net and washed with distilled water 3-7 times. Fifty larvae were placed into a 2-ml microcentrifuge tube. After adding 0.5 ml of distilled water, the larvae were grinded, the pistil was washed twice with 0.5 ml of distilled water. The extract was then filtered through a Whatman #1 filter paper (catalogue number 1001-110) and washed with a total 150 ml of distilled water. Organic solvent extracts followed a slightly different procedure. Hexane, diethyl ether, methanol, and butanol extracts were obtained in Pyrex glass homogenizers, the supernatant was filtered through Pasteur pipettes with a cotton plug, and this procedure was repeated twice. In the case of hexane and diethyl ether extracts, after www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 7. Effect of the concentration of larval extracts on oviposition stimulation. Larval extracts from 4thstage Ae. aegypti were tested in indoor assays comparing the "standard dilution" of 0.33 larvae-equivalent per ml (L-eq/ml) with lower and higher doses. N = 12. Means of the treatments were compared with the control by using the nonparametric Friedman test.

Figure 8.
Oviposition preference for larval extracts in the presence of Bacillus thuringiensis israelensis (Bti). Bti was added to traps loaded with 4th-stage larval extracts from Ae. aegypti as well as to the control water traps. Pairs of traps were deployed in the eight different locations in the field and inspected every two weeks. N = 51. Means were compared by using the Wilcoxon matched-pairs signed rank test.