Aquatain® Causes Anti-Oviposition, Egg Retention, Oocyte Melanization and Triggers Female Death in Ae. Aegypti

HAMADY DIENG (  Hamady.dieng@gov.ky ) Mosquito Research and Control Unit Storm McLean University College of the Cayman Islands Holly Stradling University of Liverpool Cole Morgan Cayman Islands Health Services Authority Malik Gordon University College of the Cayman Islands Whitney Ebanks Mosquito Research and Control Unit, Cayman Islands Zoila Ebanks Mosquito Research and Control Unit Alan Wheeler Mosquito Research and Control Unit, Cayman Islands

organochlorines [13,14], the control of Ae, aegypti with larvicides is hindered by a reduced susceptibility [15], international scrutiny and deregulation for the toxicity against biodiversity [16] and the existence of inconspicuous larval habitats [17,18].
This mosquito disperses eggs across multiple natural and arti cial containers associated with human activity [19,20,21]. Despite the multitude of potential breeding sites, Ae. aegypti has been reported to successfully locate those that can guarantee the success of the offspring and to ignore others that are unsuitable for larval development completion [22,23,24]. This ability to identify high-quality oviposition sites and select those that provide increased larval performance is mediated by many parameters, but most studies focused on the role of olfactory cues [25] and habitat physics [20]. Although there has been a focus on how some components of the site's physical structure affect egg deposition choice [26], there has been little research work regarding the physical features of water bodies, known to present several visual signals [20]. These include texture and re ectance from the water surface, which have been reported to in uence the oviposition of container-breeding mosquitoes including Ae. aegypti [27,28,29,30]. Surface tension is a property of water that plays an integral part in the development and survival of aquatic insects, including mosquitoes [31,32]. Surface tension can be generated when a water surface is covered with an oil lm [33,34]-putting oil over water results in an elastic behavior of the water, causing it to acquire a minor surface area possible [35]. Such surface tension reduction produces a lm at the airwater interface [20]. Ae. aegypti adults are incapable of resting on the water's surface when its tension is reduced [36]. Because this mosquito prefers a high level of surface tension, it exhibits high mortality responses by drowning when exposed to water with reduced surface tension [36].
In addressing dengue vector control, the World Health Organization [37] and many other mosquito scientists [38,23] have called to take oviposition into account, not only because of transovarial transmission of viruses [39] but because preventing oviposition can to disrupt the life cycle and reduce the population growth [23]. Due to these potentials positive control outcomes, some studies have been done addressing the use of oil to prevent mosquito oviposition, but most of these investigations have used either essential oils [40,41,42], lecithin monolayers [43,44,45] or agnique MMF monomolecular surface lm [46], all of which known to be easily breakable by wind and rainfall [47,48]. Although a recent study [49] has tested an oil lm with high resilience to breakages by wind and rain-Aquatain® Mosquito Formulation [50], the authors did not address oviposition and Ae. aegypti. Here, we assessed the oviposition, egg retention, oocyte melanization, and female mortality of Ae. aegypti in response to different levels of presence of Aquatain® Mosquito Formulation (AMF) in potential breeding sites.

Colony maintenance
The Ae. aegypti mosquitoes used here came from a colony maintained at the insectarium of the Mosquito Research and Control Unit (MRCU) where environmental conditions were as were 26.5 ± 1°C temperature, 65 ± 4% relative humidity, and photoperiod of 13:10 (light:dark) with 1 hour of dusk. The colony originated from larvae collected from different containers in George Town in November 2020.
Routinely, larvae were reared at a ratio of 100 to 150 larvae in plastic trays lled with 800 ml tap water. Larval food (ground Tetramin, Tetra, Germany) was supplied once daily, and the rearing medium was replaced with fresh medium once before the commencement of pupation. Pupae were placed in polystyrene cups (50-mL capacity) and transferred to BugDorm cages (30 × 30 × 30 cm, MegaView Science Co., Ltd., Taichung, Taiwan). Emerging adults had continuous access to 10% sucrose solution.
Females were arti cially offered blood meals once every two weeks. Three days after blood feeding, eggs were collected, air-dried under insectary conditions, and stored for colony maintenance or experiments.

The making of experimental subjects
To obtain gravid females, egg samples from the colony stock were submerged in tap water, and four larval population replicates, each with 200 newly hatched larvae was reared as outlined above. They were fed every two days with Tetramin. As performed for the colony, the rearing water was replaced with fresh water before the third food supply, following others [24]. Pupae were held in plastic cups and transferred into cages holding a 10% sugar solution. Three to four days old females were offered blood meals for 10 min. Fully engorged females that digested blood meals for three days were considered gravid and used as experimental subjects.

Experimental features
All oviposition tests were performed following a published experimental design [24,51] with slight adjustments. Fig. 1 shows the oviposition bioassay design. The egg deposition arena was composed of four acrylic containers (depth = 7.3 cm, diameter = 3.3 cm), each with a segment of white paper (length = 8 cm, width = 8 cm) that layered the entire surface of the container and served as an egg deposition substrate. These containers were each at a given corner of the cage, and to obviate any likely location bias, the positioning and setting of the oviposition sites was modi ed for bioassay replicate, adopting the previously described clockwise replication blueprint [24,51]. In this scheme, an oviposition test replicate accorded the layout of the four oviposition containers within the cage. All bioassay types were repeated four times, and for each replication, a batch of twelve fully blood-fed females that had digested their meals for three days, new egg deposition substrates, and new oviposition media were used. All oviposition arenas were equipped with a sugar source supplied by a cotton wick soaked with a 10% sucrose solution. The sugar supply apparatus was replaced with fresh solution and cotton wick once during the oviposition period, which lasted ten days. All oviposition bioassays were performed where temperature and relative humidity were 28 ± 2°C and 70 ± 3%, respectively.

Chemical and test concentration
Aquatain® Mosquito Formulation-AMF from (Aquatain Products Pty Ltd., Australia) containing 78% of the active ingredient polydimethylsiloxane (also called silicone), was used in this study at 1 ml/m2, the application rate recommended by the manufacturer [50]. The test oviposition medium was generated referring to the approved application rate and considering the dimensions of experimental containers used in this study. Each container used as an oviposition site has a diameter of 3.3 cm and a surface area of 33.16 cm2. Based on these dimensions, a volume of 3.316 µl of AMF was added to 150 ml of water, and the resulting solution was used as an oviposition medium. The exact nal volume in water (150 ml + 3.316 µl) served as a water medium. For convenience, containers with the test medium or water were designated as "AQ" and "W", respectively.

Bioassays
To ascertain whether or not the presence of AMF in uences the oviposition responses of Ae. aegypti, four bioassay types were run following others ( [51] Dieng et al. 2017). In the rst test, a total of twelve gravid females were placed in a BugDorm cage (30 × 30 × 30 cm) into a cage with an oviposition arena comprising four W containers (W1, W2, W3, and W4) and a sugar supply source. A second bioassay was performed as illustrated above, but the twelve females were given options to lay eggs in four AQ containers (AQ1, AQ2, AQ3, and AQ4). In the third test, the twelve females were given equal chances to lay eggs in two AMF-treated containers (AQ1 and AQ2) and two others with water (W1 and W2). In the fourth bioassay, the twelve females were presented with more options to oviposit in AMF-treated containers (AQ1, AQ2, and AQ4) than in water (W1).

Data collection and statistical analyses
At the end of the oviposition period (10 days), dead females were counted in each cage replica of each bioassay type and immediately frozen alongside survivors. Egg depositions were checked in all bioassays by procedures from Dieng et al. [51] with the help of a stereomicroscope (Motic SMZ-171-TLED; Motic Instruments Inc.; Schertz, USA). The total numbers of eggs laid in each container replica were quanti ed by counting the eggs present on the surface of the water media, paper substrates, and the lower surfaces of the bottom of the containers. The resulting numbers were used to determine the percentages of eggs laid in a given oviposition container replicate using the following formula: total number of eggs deposited in a given media divided by the total number of eggs deposited in all media of a given bioassay replicate multiplied by 100. The mean values of numbers egg deposited in a given bioassay replica by summing up all numbers of eggs in a given bioassay divided by the total number of experimental females. Percentages and mean values were utilized to score oviposition responses. Nondried females were dissected, and the two ovaries were examined for egg presence using the stereomicroscope. The numbers of eggs withheld were counted for each dissected female, and the number of eggs produced by a female was de ned as the sum of eggs laid and retained, as to follow Farjana and Tuno [52]. As did Dieng et al. [51], the egg retention rate for each dissected female was calculated as the total number of eggs withheld divided by the total of eggs produced × 100. The percentage of eggs retained by a given female was de ned as the total number of eggs retained divided by the total number of eggs produced. At each dissection occasion, the numbers of eggs that were white/light grey, dark grey with black spots, and black were recorded. We considered white/light grey, dark grey, and black colored eggs as non-melanized, melanizing and melanized, respectively. The numbers of eggs in such states were used to determine the percentages of non-melanized, melanizing, and melanized eggs. The differences in oviposition, egg retention, and eggshell melanization responses were analyzed

Results
Oviposition responses to AMF-treated water in various competition levels with water When water was the only medium, eggs were found in each of the four containers. Of the 2084 eggs laid, 30 25). Egg deposition did not differ between these containers (p = 0.862) ( Fig. 2A). When given equal chances to oviposit in two containers holding water supplemented with AMF and two others with water, Ae. aegypti females laid eggs in all containers, but oviposition responses varied appreciably with container medium.
provided with (i) matching oviposition options, (ii) more oviposition opportunities in containers supplemented with AMF, and (iii) in the AMF-based no-choice setting, respectively. The total egg deposition when water was the only oviposition medium was 1.75, 2.66, and 3.86 times higher than when half, three-fourths, and all oviposition sites were treated with AMF, respectively (Table 1). Eggs were retained in all experimental oviposition designs (Table 2), but egg retention responses varied signi cantly with oviposition arena layout (p < 0.0001). In the no-choice test involving only water, a total of 649 eggs were recovered from the ovaries of the forty-eight females ten days after blood meal uptake; this means that they retained 23.73% (649/2734) of their total egg production. In the equal-choice test, 462 eggs were collected following the dissections of the twenty-eight surviving females a week after blood digestion. When more containers were treated with the oil, 1669 eggs were retained by the forty-one surviving females. This value was 968 eggs for 38 females when containers with the oil were the only egg deposition sites. The mean number of eggs retained per female in the water-based no-choice trial (13.52 ± 3.49 eggs, range: 0 -76 eggs) was appreciably lower than that in the equal-choice experiment (17.11 ± 3.99 eggs; range: 0 -63 eggs) (p < 0.0001), which, in turn, was substantially lower than that obtained when there were more containers treated with the oil (40.70 ± 4.88 eggs, range: 0 -107 eggs; p < 0.0001). Individual females also retained arithmetically fewer eggs in the water-based no-choice compared to the AMF-based no-choice trial (25.94 ± 3.98 eggs, range: 0 -71 eggs), which in turn recorded signi cantly more eggs retained per female than the equal-choice (p < 0.0001).
Oocyte melanization patterns in responses to AMF exposure Figure 3 shows the egg melanization responses and variations after a 10-day oviposition period. When water was the only medium present in the oviposition arena, and when there was an equal chance for oviposition in two containers with water and two others with AMF-treated water, none of the females dissected had melanized eggs. Of the 1669 eggs retained by the forty-one females that were provided with more oviposition opportunities in AMF-treated containers, 76.45% (1276/1669) and 15.27% (255/1669) were partially and fully melanized, respectively. When containers treated with the oil were the only oviposition sites, 85.13% (824/968) and 14.87% (162/968) of retained eggs were moderately and fully melanized, accordingly. Numerically, the mean number of partially melanized eggs in the no-choice test involving AMF (21.68 ± 3.63 eggs) was lower than that when egg deposition options were biased towards containers supplemented with AMF (31.12 ± 4.86 eggs), but there was no signi cant discrepancy between the two mean values (p = 0.087). Females tended to have less fully melanized eggs when all sites were treated with the oil (4.26 ± 1.44 eggs) than their counterparts maintained in the arena with the sole egg-laying option in water (6.22 ± 1.40 eggs), but the difference was insigni cant (p = 0.092).

Mortality responses of Ae. aegypti females during oviposition in AMF presence
There were signi cant differences in the survival of Ae. aegypti females between the different oviposition opportunities (p < 0.0001). When containers with water were the only egg deposition sites, the mean female mortality was 4.16 ± 4.16%, and oscillated between 0 and 16.66%). Arithmetically, this latter mean was far lower than that recorded when females were given equal choices to oviposit in containers with AMF-treated water and two others with water (16.66 ± 4.16%); however, the difference was insigni cant (p = 0.539). The mean mortality rate of females in the "more AMF options" experiment (42.833 ± 7.21%, range: 25 -58.33%) was signi cantly greater than that of the "equal-choice experiment (p = 0.034), which in turn, was numerically lower that from the AMF-based no-choice test. There was no signi cant difference in female death when there were more options in containers holding AMF-treated water and when only containers with AMF-treated water were present (p = 0.901) (Fig. 5).

Discussion
The current study revealed signi cant adverse impacts of the label dose of the silicone-based monomolecular lm on Ae. aegypti. Oviposition responses were de cient as the presence of containers with the oil increased. Egg retention and internal oocyte melanization rates were high in oviposition arenas dominated by the presence of containers with the oil; such effects decreased in water presence, especially under water-based no-choice conditions. Death occurred at greater rates among females maintained in arenas with the increased presence of oviposition sites treated with the oil compared to arenas with elevated water presence.
The appeal or repulsiveness of competitive egg deposition sites to gravid mosquito females is dictated by a diversity of factors, among which the most important is physico-chemistry of the water media [54] and the prospect of unfavorable conditions for larval development completion [55,20]. Various physical elements have been evidenced to prevent egg deposition in mosquitoes [20,56,57]. In particular, water surface tension has been shown as a critical factor in uencing oviposition [22]. Water tension typically occurs when oil is added to water; following addition, the layer of water molecules and that of oil molecules balance force between the two interfaces, causing the reduction of the surface tension at the water surface [35] and the apparition of a lm [32]. Ae. aegypti has been reported to select oviposition sites concerning the possibility of larval development completion [55,22,23,24]. In mosquitoes, the presence of an oil lm on the surface of the water of potential breeding habitats has often been associated with reduced survival. For instance, Mbare et al. [50], dealing with Anopheles vectors, noted a 90% larval mortality and over 80% adult emergence inhibition with an AMF lm. Dawood et al. [32], working with Culex pipiens, investigated the effects of AMF on different immature stages. They found signi cantly far greater mortality rates in rst-, second-and fourth-instar larvae and pupae in treated containers than untreated ones; they attributed these impacts to the ability of AMF to alter the water surface tension. In a recent study, Kavran et al. [49] examined the lethality of AMF on the immature forms of two mosquito species and reported higher mortality rates of the juveniles of the dengue vector, Ae. albopictus. In an earlier study, Ngrenngarmlert et al. [58] assessed the lethal potential of a silicone-based monomolecular lm against Ae. aegypti and reported signi cant larval and pupal mortality rates. Besides the immature stages, the presence of oil can affect the behaviors of adults [48], including ovipositing females [43]. The mechanism by which this phenomenon occurs has been well documented. According to Likura et al. [59], mosquito legs are highly hydrophobic, and this trait causes a weight-supporting force on water surfaces. For a successful egg-laying to occur, a gravid female must generate a repulsive force from to legs to be able to use the surface of the water as a foothold [58,59] and evaluate its quality [20]. However, when silicone oil is added over the water surface, it generates an attractive capillary force that drags legs towards the water [58], inducing a short contact time and an escape response [59]. A shortened contact time and startle will result in an incomplete exploration of the quality of the oviposition site and thus a decreased probability of egg deposition. The current study was carried with two colorless media oviposition, i.e., AMF-treated water (150 ml of water + 3.316 µl of AMF) and water (150 ml + 3.316 µl). All the containers used in oviposition bioassays had a similar con guration, and no food was added to any container; as such, differences in oviposition responses due to difference in con guration is unlikely. Based on the reports mentioned earlier and our methodology, the observed low oviposition responses in AMF-treated containers could be explained by an attempt of the gravid females to maximize survival for both offspring and themselves. It is possible that the ovipositing Ae. aegypti females associated AMF presence with poor nutritional quality and reduced chances of larval development completion. It is also likely that AMF has reduced the water surface tension, which in turn has prevented gravid females from either landing or staying long enough on the surfaces of near container edges to release their eggs.
The egg retention rates in Ae. aegypti females placed in environments dominated by the presence of containers treated with AMF were far greater than that of females kept in arenas with only water as oviposition medium. Several studies have reported egg retention in mosquitoes relative to oviposition site features. Chadee et al. [60], working with the species studied here, increased egg retention behavior seven day-post blood meal when females were not provided with egg-laying substrates.
Dealing with a dengue vector, Satho et al. [24] assessed changes in its oviposition responses in no-choice and choice bioassay involving different coffee extract concentrations. They observed that Ae. albopictus females withheld increased numbers of mature eggs when cups with highly concentrated extract were the only available sites. Seenivasagan et al. [23] reported that the topical repellent diethyl phenylacetamide deterred egg depositions by the Ae. aegypti females, which retained around half of their egg productions. Xue  Although we did not assess the effects mentioned above, our study revealed some information on melanization, known to play various crucial roles in insects [68]. There was a clear link between oviposition medium type and eggshell melanization level. No oocyte melanization occurred when gravid females were exposed to water or AMF-treated water in balanced competition with water contained. In contrast, egg groups dissected from the ovaries of females exposed to an increased presence of AMFtreated water showed a high prevalence of melanized eggs, those from arenas holding exclusively AMFtreated water, where all retained eggs exhibited different levels of melanization (whitish/Light grey-nonmelanized, dark grey with black spots-partially melanized and black-fully melanized). In dengue vectors, this biochemical process [69] generally starts after oviposition [70,71]. Newly laid eggs that are whitish and smooth [72] undergo a melanization/sclerotization process [73] to become gradually black and hard after two to four hours [72,74,75]. During the process, a cascade of enzymatic synthesis and activities involving the phenoloxidase [75] and DOPA decarboxylase [76,70] lead to the production of melanin. Recently, Isoe et al. [71] assessed the genetic aspects of melanogenesis in Ae. aegypti, and found that the eggshell organizing factor 1 (EOF1) plays an essential role in melanization. They suggested that partial melanization prior to oviposition is caused by the loss of EOF1 activity, which alters the chemical balance within the oocytes, which in turn activates other eggshell components to initiate the melanization process prematurely. They also noted that EOF1-de cient mosquitoes have eggs with different melanization levels, varying from non-melanized to completely melanized eggshells. In the current study, gravid Ae. aegypti females were presented with AMF-treated water as oviposition medium in different competition levels with water for the same period (10 days). Although we did not investigate these phenomena, contingent on the elucidations from the studies quoted above, it is presumable that the synthesis and levels of the phenoloxidase and decarboxylase were high in the eggs of females exposed solely to AMF-treated water and when three-fourths of the oviposition containers were treated with AMF. It is plausible that the observed disparities in melanization levels between AMF-treated water-exposed females and non-exposed or less exposed females ensued due to the high activity levels of these enzymes. Such effect of AMF treatment on the activities of phenoloxidase and decarboxylase was possibly less marked in females with no (water treatment) or reduced exposure (equal-choice and threefourths treatments) to AMF. It is also conceivable that the females exposed solely to AMF-treated water had more EOF1 expression levels and activities. During the 10-day oviposition period, the mortality rates of gravid females were high in arenas with the increased presence of AMF-treated oviposition containers, especially the "AMF-based no-choice treatment", compared to water-based no-choice treatment", thus denoting the presence of adulticidal factors of the AMF medium. AMF is a polydimethylsiloxane (PDMS, 80%)-based liquid that is not toxic [32] as indicated by its certi cation for use in drinking water [77,78]; so, death due to toxic chemicals is unexpected. The observed differences in female mortality responses may have been due to at least two causes. First, reducing the water surface tension in the AMF-treated containers, particularly in the AMF-based no-choice and three-fourths treatments, may have produced strong, attractive capillary forces that dragged the legs of females towards the water, consequently increasing the likelihood of sinking and death. In support of this assertion, on the water surface, but not on the water surface with a silicone oil lm, female mosquitoes can use the surface as a foothold to lay their eggs, using a maximal repulsive force from their legs [59]. Second, the untimely internal oocyte melanogenesis during egg retention may have occasioned the production and accumulation of toxic compounds among females with increased exposure to AMF-treated water, which elevated the possibility of intoxication. In favor of this contention, it has been shown that phenoloxidase activity in the insect hemolymph can produce quinonoid toxic compounds [79] and that melanin and its quinone intermediates are toxic to hosts [70].

Conclusions
We observed that treating all, three-fourths or half of potential breeding sites of Ae. aegypti in a given environment with AMF altered its potential contribution to the next generation. AMF treatment prevented females from laying sizeable numbers of eggs, caused increased egg retention and internal egg melanogenesis, which are conducive to reproductive unsuccess. AMF inoculation in potential developmental sites also triggered the death of gravid females, thus denoting adulticidal potentials.
Evidently, AMF performed as an oviposition deterrent and as an indirect killer of ovipositing A. aegypti females; this latter observation would be a supplemental bene t to vector control programs. These attributes, coupled with its previously recognized direct lethality on the immature stages of dengue vectors [58,49], advocate for the incorporation of AMF into integrated approaches for dengue vector control. As AMF is safe for humans [77,78,50], its use in domestic areas where Ae. aegypti thrives naturally, may be widely accepted. In addition, as AMF's mode of action is physical, its use and rotation with other larvicides can help mitigate insecticide resistance issues. Figure 1 Oviposition bioassay design. The containers were placed on the dish, which was positioned at the bottom center of the cage. Containers were placed such that each was at an identical distance from the adjacent container. A bioassay replicate coincided with one arrangement of the four containers. Melanization response patterns of the oocytes of gravid Ae. aegypti females after a 10-day exposure to Aquatain® Mosquito Formulation-(AMF)-treated water in various competition levels with water. A: Whitish/light grey eggs (non-melanized) when water was the only medium present in the oviposition arena (water-based no-choice) and when there was an equal chance to oviposit in two containers with water and two others with AMF-treated water (Equal choice); B: Dark grey with black spots (melanizing or partially melanized) when AMF-treated water was the only medium present in the oviposition arena (water-based no-choice); and C: Black eggs (fully melanized) in AMF-based no-choice bioassay.