Journal of Medical Entomology, XX(XX), 2022, 1–9
https://doi.org/10.1093/jme/tjac175
Research

Vector Control, Pest Management, Resistance, Repellents
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Hydroxyapatite-binding Silver/Titanium Dioxide as a
Potential Control Compound Against Mosquito Vectors,
Aedes aegypti (Diptera: Culicidae) and Anopheles dirus(Diptera: Culicidae)
Raweewan Srisawat,1,7, Patchara Sriwichai,1 Jiraporn Ruangsittichai,1
Chawarat Rotejanaprasert,2,3 Naoko Imaizumi,4 Dai Yamaki,4 Maki Maekawa,5Yuki Eshita,6,7, and Narumi Okazaki4
Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok 10400, Thailand,
Department of Tropical Hygiene, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok 10400, Thailand,
3
Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok 10400,
Thailand, 4DR.C Medical Medicine Co., Ltd., Shinjuku, Tokyo 160-0023, Japan, 5Seltec Co., Ltd., Hachioji, Tokyo 192-0062, Japan,
6International Institute for Zoonosis Control, Hokkaido University, Sapporo, Hokkaido 001-0020, Japan, and 7Corresponding authors,e-mail: raweewan.sri@mahidol.ac.th (R.S.), yeshita@czc.hokudai.ac.jp (Y.E.)
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Subject Editor: Ary Faraji
Received 26 May 2022; Editorial decision 18 October 2022.

Abstract
Controlling mosquitoes is vital for counteracting the rising number of mosquito-borne illnesses. Vector control requires the implementation of various measures; however, current methods lack complete effectiveness,
and new control agents or substances are urgently needed. Therefore, this study developed a nonwoven
fabric sheet coated with hydroxyapatite-binding silver/titanium dioxide compound (hydroxyapatite-binding
silver/titanium dioxide sheet [HATS])and evaluated its effectiveness on all stages of laboratory Aedes aegypti(Linnaeus); Diptera: Culicidae and Anopheles dirus (Peyton & Harrison); Diptera: Culicidae. We reared larvae
with HATS and control sheets and assessed their mortality, emergence, and hatching rates. The submersion
rates of engorged female mosquitoes in submerged HATS and control sheets were also compared. The HATS
strongly affected mosquito development, resulting in high mortality rates (mean ± SE) of 99.66 ± 0.58% (L1–L2)
and 91.11 ± 9.20% (L3–L4) for Ae. aegypti and 100% of both stages for An. dirus. In contrast, mosquitoes raisedin the control sheet showed relatively high survival rates of 92.33 ± 3.21% (L1–L2) and 95.67 ± 0.58% (L3–L4)
for Ae. aegypti and 86.07 ± 3.53% (L1–L2) and 92.01 ± 8.67% (L3–L4) for An. dirus. Submersion of engorgedfemales was found in the HATS oviposition cup, leading to a decreased number of eggs and a low hatchingrate compared to that of the control. Overall, HATS may be a useful new control method for Ae. aegypti andAn. dirus.
Key words: vector control, hydroxyapatite-binding silver/titanium dioxide, mortality, submersion rate, hatching rate

© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Journal of Medical Entomology, 2022, Vol. XX, No. XX

Graphical Abstract

metal nanoparticles have received substantial attention owing totheir desirable characteristics, such as inexpensiveness, having a wide
range of applications, excellent antimicrobial activity, less toxicity tohumans, ecofriendliness (Santhosh et al. 2015, Kumar et al. 2020,
Narayanan et al. 2021), and larvicidal effect against mosquitoes(Patil et al. 2012, Kumar et al. 2017, Pilaquinga et al. 2019). Silveris the most popular metal for the synthesis of nanoparticles (AgNPs)
from plant extracts for use in insect vector control, and thosegenerated from Avicennia marina (Barnawi et al. 2019), Annonasquamosa (Arjunan et al. 2012), Lippia citriodora (Elemike et al.
2017), Derris trifoliata (Kumar et al. 2017), Leucas aspera, andHyptis suaveolens (Elumalai et al. 2017) exhibit mosquitocidal effect against Ae. aegypti, An. stephensi Liston (Diptera: Culicidae),and Culex quinquefasciatus Say (Diptera: Culicidae).
The fabrication of titanium dioxide (TiO2) has received increased
attention recently owing to its versatile applications, such as in healthcare products (sunscreen lotions, beauty creams, skin ointments, etc.)
and electronic industries (capacitors, electrochemical electrodes,
solar cells, etc.) (Kamaraj et al. 2010). The TiO2NPs prepared fromthe plant extracts of Pouteria campechiana (Narayanan et al. 2021),
Parthenium hysterophorus (Thandapani et al. 2018), Ficus religiosa(Soni and Dhiman 2020), and Morinda citrifolia (Suman et al. 2015)
exhibited larvicidal effects against mosquitoes. These plant extracts
reduced and stabilized titanium as nanoparticles. Green TiO2NPs
have a higher toxicity than plant extracts alone.
A hydroxyapatite-binding silver/titanium dioxide compound(produced by DR.C Medical Medicine Co., Ltd.) has been proposed
as a new mosquito control chemical (DR.C Medical Medicine Co.,
Ltd. n.d.-a). It was first developed as an innovative treatment for
sheets and masks that protect against pollen allergies and was later
used by several Japanese companies to manufacture items, such as
shirts, socks, and towels, that prevent and delay bacterial growth(DR.C Medical Medicine Co., Ltd. n.d.-b). Although the apatiteTiO2-coated cotton fabric can decompose antigenic proteins, bacteria, pollen, mold, and viruses (Okazaki et al. 2022), its ability

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The mosquito is one of the most dangerous insects in the world(Daniel and Wunderman 2020). Infected mosquitoes can carryand spread diseases, such as dengue fever and malaria to humans
through bites. Dengue fever has recently become more widespread
globally (WHO 2021). Currently, approximately half of the humanglobal population has a risk of contracting dengue. The first outbreak of dengue hemorrhagic fever occurred in Thailand in 1958
(Rojanapithayakorn 1998), and its prevalence has undergone a cyclic increase. The principal dengue vector in Thailand, Aedes aegypti(Linnaeus; Diptera: Culicidae), was originally reported in 1907
(Theobald) and is also an arbovirus vector of Zika yellow fever, andchikungunya virus (WHO 2021). It is a daytime biting mosquito,
with peak bite periods in the early morning and before dusk, and amultiple feeder, allowing it to spread mosquito-borne diseases more
efficiently (Trpis et al. 1973). The eggs laid by a female can persist forseveral months before hatching, which occurs during contact withwater.
One of Thailand’s major malaria vectors is Anopheles dirus(Peyton & Harrison); Diptera: Culicidae, which is found all overThailand. It feeds between sunset and midnight, a behavior known
as exophagic and anthrophilic (Tainchum et al. 2015, Tananchai
et al. 2019). Owing to the increasing number of mosquito-borne
illnesses and the lack of a completely effective vaccine, the prevention of these diseases is primarily achieved through effective mosquito control (WHO 2009, Amarasinghe et al. 2020).
On the one hand, chemical control using insecticides has beensuccessful in controlling the target vector species, such as Aedes(Srisawat et al. 2010) and Anopheles (Corbel et al. 2013). On theother hand, mosquitoes are increasingly becoming less susceptible
and developing resistance to chemical insecticides (Hemingway et
al. 2004, Dusfour et al. 2019, Duarte et al. 2020), resulting in a rebound in vectorial capacity (Elumalai et al. 2017). Therefore, it isrequired to identify an agent that reduces the selection pressure of
insecticide resistance and is easily degradable, safe, and environmentally friendly (Manjarres-Suarez and Olivero-Verbel 2013). Recently,

Journal of Medical Entomology, 2022, Vol. XX, No. XX

3(HATS). Two control sheets were used: a light blue nonwoven fabriccontaining only a defoamer and phthalocyanine blue coloring (original color before compound treatment; blue sheet control, BC) anda non-woven fabric sheet of white color (white sheet control, WC)(Fig. 1).

Mosquitoes

to kill mosquitoes has not yet been investigated. Only the application of AgNPs coated with natural active ingredients that killmosquitoes has been reported. There have been several reports
of using AgNPs to kill mosquitoes at all life stages. Balakrishnan
et al. (2016) used positively synthesized AgNPs from mangrove
plants as a larvicide against Ae. aegypti and An. stephensi, andShanmugam et al. (2014) assessed the effectiveness of positively
green-synthesized AgNPs as a larvicide and pupacide against these
two mosquito species. Furthermore, Murugan et al. (2016) reported the use of synthesized TiO2NPs as a larvicide and pupacide
against Ae. aegypti.
The compound is a catalytic substance of anatase-type titaniumoxide with added silver that uses hydroxyapatite. The titanium dioxide is a photocatalytic substance that can break down organic
compounds, microbial organisms, such as viruses and bacteria, andcancer cells. It has been used for the sterilization of medical devices,
food preparation surfaces, air conditioning filters, and sanitary-waresurfaces (Okazaki and Hoshi 2016). To functionalize the compound,TiO2 is irradiated with energy greater than its band gap energy, andan electron is excited from the valence band to the conduction band.
Therefore, electron-hole pairs are formed that react with water or oxygen molecules to form various reactive oxygen species (ROS). The titanium dioxide with additional silver exhibits efficient photocatalytic
properties and antibacterial activity. Hydroxyapatite absorbs bacteria,
microbes, pollen, mold, proteins, and viruses, which are then broken
down by free radicals of the TiO2 photocatalyst (Taoda 2009).
In preliminary studies, the compound was able to kill mosquito
larvae and is, therefore, a promising larvicidal agent. However, its
safety is an important concern. The HATS has been registered with
the Pharmaceuticals and Medical Devices Agency (PMDA, https://www.pmda.go.jp/english/index.html) in Japan and was confirmed
to be a safe product for humans and animals (Okazaki and Hoshi
2016), which are vital properties for a vector control agent. Herein,
we evaluated the effectiveness of HATS against mosquitoes.

Materials and Methods
HATS
A dark blue nonwoven fabric sheet of 1 × 1 m (DR.C Medical
Medicine Co., Ltd.) containing 13.5 g of HATS, defoamer, andphthalocyanine blue color was used as the experimental sheet

Early-stage Mosquito Larvae Mosquito eggs were immersed in a
250-mL cup of reverse osmosis (RO) water (100 mL) lined with a
HATS, BC, or WC sheet for one to two days. The hatched larvae(L1-L2) were used as early-stage larvae in this study.
Late-stage Mosquito Larvae Eggs hatched in 100 ml RO water wereallowed to develop into L3–L4 larvae for use in the late-stage study.
The larvae were fed daily with fish food (Optimum Hi Pro Growthand Color) and maintained at 25 ± 2°C until they reached L3–L4
stage.

Effect of HATS on the Early- (L1–L2) and Late-stage(L3–L4) of Ae. aegypti and An. dirus Larvae
RO water (100 ml) was added to a 250-ml plastic cup(9.2 × 7.6 × 5.5 cm top diameter, bottom diameter, and height, respectively, #PC250 MB, FP Chupa Corporation, Tokyo, Japan) witha 2 cm high water column, 56.72 cm2 surface area, and 45.34 cm2
bottom surface area. Sections (11 × 11 cm) of HATS, BC, andWC sheets were submerged in the bottom of separate cups. There
was 0.1635 g of hydroxyapatite-binding silver/titanium dioxide
compound particles (=0.11 × 0.11 m × 13.5 g/m2). For each mosquito stage, 20 larvae were added to each experimental and control cup, with five cups per treatment. The entire experiment was
replicated three times. The larvae were reared at 25 ± 2°C with
0.0046 ± 0.0008 g of larval food provided daily (fish food: Optimum
Hi Pro Growth and Color) until they developed into adults. The
daily number of larvae, pupae, and adults in each cup was recorded,
and the survival rate was calculated using the following formula:
Survival rate = (number of live larvae, pupae, or adult/number of larvae tested) × 100

Effect of HATS on Submersion of Female
Mosquitoes and Egg Hatching for Ae. aegypti andAn. dirus
Oviposition Cup For An. dirus, three 250-ml plastic oviposition cups
were used containing 80 ml of RO water and four pieces (5.5 × 5.5 cm)of the sheets (HATS, BC, WC) submerged in the bottom of each cup.
For Ae. aegypti, one (11 × 11 cm) and two pieces (5.5 × 5.5 cm) of
each sheet were submerged in three oviposition cups. The number ofsheets was verified from our preliminary tests (unpublished data)—toDownloaded from https://academic.oup.com/jme/advance-article/doi/10.1093/jme/tjac175/6827101 by guest on 28 November 2022

Fig. 1. Three types of sheets used in this study. Left – a non-woven fabricsheet of 1 × 1 m containing 13.5 g of hydroxyapatite-binding silver/titanium
dioxide compound, defoamer, and phthalocyanine blue (HATS). Middle – anonwoven fabric containing only defoamer and phthalocyanine blue (BC).
Right – nonwoven fabric sheet (WC). Abbreviations: HATS, hydroxyapatitebinding silver/titanium dioxide sheet; BC, blue sheet control; WC, white sheet
control.

Laboratory colonies of Ae. aegypti (Bora Bora strain) and An.
dirus (Khao Mai Kaew strain) were used in this study. To maintainmosquitoes in the laboratory, eggs were hatched in plastic trays(20 × 30 × 5 cm). After hatching, 200 larvae were reared in plastic
trays with 1,500 ml of dechlorinated water and fed fish food powder
daily (Optimum Hi Pro Growth and Color, Perfect Companion
Group, Bangkok, Thailand). Pupae were transferred to plastic cups
and placed in a cage (20 × 20 × 30 cm) until emergence. Adult
mosquitoes were fed 5% sugar solution on cotton wool, which waschanged weekly. They were reared and maintained at 25 ± 2°C and
65 ± 10% relative humidity at the insectarium in the Departmentof Medical Entomology, Faculty of Tropical Medicine, Mahidol
University.

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4determine the number of HATS that affect mosquito egg hatchingrates and adult submergence rates. The tests suggested that Ae. aegypti
required approximately 1.5 times the number of HATS.
Submersion of Female Mosquitoes Thirty fully blood-fed inseminatedfemale mosquitoes, at 4 d post-blood feeding, were released into
each cage. The individual oviposition cups were transferred to each
adult mosquito cage, and three cages were used for each species. The
female mosquitoes were maintained for 3 d. The total number offemales that submerged and laid eggs was recorded after three daysof exposure. Three replicates were performed.
Egg Hatching An. dirus eggs laid on the sheet were maintained in thesame cup until they hatched. Ae. aegypti eggs were dried at 27–29°C
for 3 d and then submerged in the cup. The number of hatchedlarvae was counted for one week.

Data Analysis
Continuous data from each experiment were tested for normal distribution. The non-normally distributed data were analyzed using nonparametric tests. We evaluated the differences among the sheets using
the Kruskal–Wallis test of one way analysis of variance (Kruskal andWallis 1952). For the survival rate and submersion proportions, themulti-sample test for equality of proportions with chi-square test
was applied, followed by pairwise comparison using Bonferroni
correction. Statistical significance was set at p < 0.05 and analyzed
using PASW for Windows version 18.0 and R version 1.4.1717. The
proportions were calculated using the following formulas:
Egg hatching rate = (number of larvae hatched/number of eggs oviposited) × 100

Submersion proportion of adult mosquitoes = total number ofadult mosquitoes submerged/number of adults tested
Survival rate = (number of live larvae, pupae, or adult/number of larvae tested) × 100

Results
Effect of HATS on Early- (L1–L2) and Late-stage (L3–
L4) Mosquito Larvae
Aedes aegypti When early-stage larvae (L1-L2) of Ae. aegypti werestored in containers with HATS, BC, and WC sheets, there weresignificant differences in the survival proportions among sheet types,
with the highest in WC and the lowest in HATS containers (Fig.
2a) (Kruskal-Wallis, H = 40.232, df = 2, p < 0.0001). The survival
proportion of larvae from the HATS group, i.e., the percentage thatsuccessfully developed to the adult stage, was only 0.34%, whereasthat of the L3–L4 larvae, pupae, and adults gradually decreased to18%, 16%, and 14%, respectively, in the BC group. Most early-stage
larvae (L1–L2) in the WC container developed into adults (93%)(Fig. 2a).
When the late-stage larvae (L3–L4) of Ae. aegypti weremaintained in a HATS, BC, or WC sheet container, the final survival proportions in HATS (9% at pupal stage, 9% at adult stage)
were significantly different from those in the WC (96% at pupal
stage, 96% at adult stage) and BC (52% at pupal stage, 50% at adultstage) containers (Fig. 2b) (Kruskal–Wallis, H = 39.024, df = 2, p <0.0001). Early-stage Ae. aegypti larvae were more sensitive to HATS
than late-stage Ae. aegypti larvae, and HATS had a strong lethal effect on Ae. aegypti of both stages.
Anopheles dirus No surviving pupal or adult An. dirus wereobserved in the HATS containers of early-stage (L1–L2) or late-stage(L3–L4) larvae. In the BC sheet containers, some early-stage larvae
developed into pupae (14%) and adults (14%) (Fig. 3a), but almost
none of the late-stage larvae developed further (Fig. 3b). It was moredifficult for the late-stage An. dirus living in HATS and BC sheet
containers to develop into pupae and adults than for the early-stage
larvae. However, the survival percentages in HATS and BC weredifferent from those in the WC (Kruskal–Wallis, H = 41.862, df =2, p < 0.0001).

Developing Time
The days taken to develop from early-stage larvae (L1–L2) into
adults of the surviving Ae. aegypti were significantly different between the types of sheets (Kruskal–Wallis, H = 559.52, p < 0.0001).
The late-stage larval (L3–L4) to adult development in the HATS
group was also significantly different from the development daysof the BC and control cup groups (Kruskal–Wallis, H = 341.30, p< 0.0001) (Fig. 4). Because no An. dirus early- or late-stage larvae

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Fig. 2. Survival of Ae. aegypti after exposure to HATS, BC, or WC. a) Survival ratios of L3–L4, pupal, and adult stages after exposure to the sheets as early-stage
larvae (L1–L2). b) Survival ratios of pupal and adult stages after exposure to the sheets as late-stage larvae (L3–L4). The error bars indicate the standard error.
Abbreviations: Refer to Fig. 1 on HATS, BC, and WC; RO, reverse osmosis water.

Journal of Medical Entomology, 2022, Vol. XX, No. XX

5survived in the HATS container, the lifecycle was not completed,and the development time could not be calculated. Moreover, thedevelopment time of early-stage An. dirus was significantly different between the BC and WC containers (Kruskal–Wallis, H =472.178, p < 0.0001) (Fig. 4). Both were significantly different
from that of the HATS container. Anopheles dirus late-stage larvae
did not survive in the HATS and BC containers (not significant);
however, that in both (HATS and BC) was significantly different
from that in the WC container (Kruskal–Wallis, H = 767.244, p< 0.0001).
Altogether, exposure to HATS of the two mosquito species
resulted in the death of almost all early-stage larvae (<1% survival rate). Furthermore, it had more effect on the survival rate oflate-stage An. dirus than on that of Ae. aegypti. The survival ratesof HATS-exposed larvae were significantly different from thoseof the BC and WC control larvae. However, it had a greater influence on the survival rate of late-stage An. dirus than on that ofearly-stage An. dirus. Early- and late-stage An. dirus larvae wereboth sensitive to the HATS compared to Ae. aegypti, of which
only the early-stage larvae were sensitive. Subsequently, the HATS
affected the early-stage more than the late-stage of Ae. aegypti
larvae, which showed a low survival rate and increased time toadult emergence.

Effect of HATS on Submersion of Adult Mosquitoes,
Egg Hatching, and Number of Eggs Laid
Aedes aegypti The submersion of blood-fed females of Ae. aegypti
submerged with HATS (6.67%) and BC sheets (5.56%) was foundin the oviposition cups; however, a similar phenomenon was notobserved in the WC control (Fig. 5a). The fewest eggs were laid in
the HATS group (Table 1). Furthermore, the hatching rates (Fig. 5b)
were significantly different (chi-square = 889.6, df = 2, p < 0.0001)
among the eggs exposed to the HATS (43.11%), BC (52.32%), andWC control (79.35%) (Fig. 5b).
Anopheles dirus The submersion rate of An. dirus females in watercontaining the HATS (58.89%) was significantly higher than thatof the WC control (1.11%) with the white sheet (Fig. 5a), and thetotal number of eggs laid was different among the groups (Table 1).
There was no difference in submersion, but there was a differencein hatching rates between the HATS and BC sheet (chi-square =687.95, p < 0.0001). However, the 29.59% hatching rate of eggs
oviposited in HATS was significantly different (chi-square = 1321.5,
p < 0.0001) from that in the WC control (73.41%) sheet conditions(Fig. 5b).

Discussion
The HATS is composed primarily of TiO2, silver, and apatite. Apatite
can absorb bacteria and fungi; adding silver to TiO2 improves its
catalytic efficacy and antibacterial activity by absorbing not only ultraviolet light (below 380 nm) but also visible light in the range of
700–800 nm (Durango-Giraldo et al. 2019, Okazaki et al. 2022).
Similarly, apatite-TiO2-coated cotton fabric used in the study by
Kangwansupamonkon et al. (2009) showed antibacterial activity
against Staphylococcus aureus, Escherichia coli, and Micrococcus
luteus. The apatite-coated TiO2 was irradiated and activated. It was
proposed that the killing effect was mediated by ROS, such as OH−,
O2−, and H2O, generated on the irradiated TiO2 surface, which may
attack and decompose polyunsaturated phospholipids in bacteria(Maness et al. 1999). The ROS production may break down the cellwall and outer membrane, causing leakage of the cell content andresulting in cell irregularities and depressions (Kangwansupamonkon
et al. 2009).
In this study, the hydroxyapatite-binding silver/titanium dioxide
compound particle was used to treat nonwoven fabric sheets (HATS)
at 0.1635 g of 0.0121 m2 sheet, along with a defoamer, and phthalocyanine blue color. The compound-treated sheet (HATS; developed by
DR.C Medical Medicine Co., Ltd. in Tokyo, Japan; https://drciyaku.
jp/mechanism.html) was lethal to larval mosquitoes. The amount used
may be sufficient to kill the larvae of Ae. aegypti and An. dirus laboratory strains; however, questions remain regarding its applicability
in the field. Not only did 0.1635 g of the compound kill larval Ae.
aegypti and An. dirus, but the defoamer and phthalocyanine blue
color also destroyed the larvae, as the larvae maintained with the BC(noncompound-treated sheet) showed a lower survival rate. The BC

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Fig. 3. Survival of An. dirus after exposure to HATS, BC, or WC. a) Survival ratios of L3–L4, pupal, or adult stages after exposure to the sheets as early-stage
larvae (L1–L2). b) Survival ratios of pupal and adult stages after exposure to the sheets as late-stage larvae (L3–L4). The error bars indicate the standard error.
Abbreviations: Refer to Fig. 1 on HATS, BC, and WC; RO, reverse osmosis water.

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Journal of Medical Entomology, 2022, Vol. XX, No. XX

sheet also affected early-stage larvae and had a marked effect on latestage An. dirus. Both the HATS and BC sheets contained defoamer andblue phthalocyanine color, whereas the WC sheet did not. We have no
explanations for these phenomena; however, these types of chemicals
may cause larval mortality owing to synergistic effects. Although BC
could kill the larvae, it was less effective than HATS, which killed alllarvae. The important thing regarding BC and HATS was the difference in hatching ability; this was especially noted in An. dirus.
There seemed to be a delay in the time to complete the lifecycle
for the larvae of both species that survived under HATS and BC
conditions, although most larvae perished, especially those in the
HATS containers. The submersion rate of An. dirus gravid females
in water containing the HATS was higher than that of Ae. aegypti
under the same conditions. This difference is likely attributed to thetypical oviposition habits of each species. Anopheles dirus females
lay eggs on the surface of the water, whereas Ae. aegypti females
primarily lay eggs on the water’s edge. Similar to water striders,mosquitoes can usually float on the surface of the water. Since water
striders float owing to surface tension, HATS may impair the surface tension of water. Finally, we noted that An. dirus was more sensitive to HATS than Ae. aegypti, resulting in lower egg hatching,

lower survival, and higher submersion rates. Therefore, the killing
effects of HATS appeared to be different for each mosquito species.
The amount of HATS needed for a killing effect should be adjusted
for each species. Furthermore, the hatching rate of eggs exposed to
the HATS was significantly different from those of the WC control
group for both Ae. aegypti and An. dirus.
The mechanisms by which HATS acts on Ae. aegypti and An.
dirus are unclear. Silver and TiO2 have antibacterial effects (Tuduet al. 2020); however, the relationship between their antibacterial
effects and lethal effects on mosquitoes is unknown. The compound
may penetrate the intracellular space and then either bind to sulfur
from proteins or phosphorus from DNA, which in turn leads to denaturation of organelles, enzymes, and finally reduced ATP synthesis,
similar to the mechanism behind the effect of AgNPs on Ae. aegypti(Skorb et al. 2008, Sundaravadivelan et al. 2013).
Although it is unknown exactly how ROS produced by thehydroxyapatite-binding silver/titanium dioxide compound affectmosquito larvae, ROS caused membrane damage in Ae. aegypti
larvae under conditions of oxidative stress (Costa et al. 2022)
and low egg production and poor developmental in Drosophilamelanogaster (Diptera: Drosophilidae) (Philbrook et al. 2011).

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Fig. 4. Development duration of surviving mosquitoes from larvae to adult. Time of development (d) from surviving larvae to adult stage Ae. aegypti (top) and
An. dirus (bottom) when exposed to HATS, BC, or WC sheets as early- (left) or late-stage larvae (right). ****P < 0.0001; NS, not significant, analyzed by Kruskal–
Wallis test. Abbreviations: Refer to Fig. 1 on HATS, BC, and WC; RO, reverse osmosis water.

Journal of Medical Entomology, 2022, Vol. XX, No. XX

7

Table 1. Number of eggs laid by engorged female Ae. aegypti andAn. dirus in oviposited cups
Species

N

HATS

BC

WC

Ae. aegypti
An. dirus

90
90

661
1,936

2,905
3,311

6,143
8,222

Abbreviations: HATS, hydroxyapatite-binding silver/titanium dioxide
sheet; BC, blue sheet control; WC, white sheet control.

On the other hand, TiO2 can regulate a material’s surface’s
wettability. The surface turns from hydrophobic to hydrophilic when
exposed to UV light (Wang et al. 1997). The hydrophobic surfaceof mosquitoes makes them water resistant. However, HATS-treatedwater was shown to hydrophilically cling to the surface in our unpublished observation. Thus, we speculated that adult mosquitoes perish
by being submerged into the water owing to the hydrophilizationof their legs, which land on the surface of the HATS-treated water.
The surface of fresh mosquito eggs laid on plain water changes
from milky white to black and forms hard shells that are impermeable to the water (Farnesi et al. 2015). However, whether TiO2 isinvolved in the formation of this shell is unknown. The surface ofeggs laid in HATS-treated water turned black. However, infiltrationof the treated water into the eggs may continue owing to the hydrophilic properties of TiO2.
The limitations of this study are that the half-maximal effective
concentration (EC50) and the precise mechanism of the killing of effect of HATS were not elucidated, warranting further studies. EC50 isoften used to evaluate mosquito mortality using juvenile hormonelike insecticides. When evaluating the lethal effect of HATS on
mosquitoes, it is necessary to consider values such as the number ofadults submerged in water, the number of unhatched larvae, and thenumber of abnormally emerged adults.
In conclusion, we demonstrated that HATS is a potential new
mosquito control measure, especially against Ae. aegypti, when it is
attached to the water container. It prevented mosquitoes to lay eggs
by deterring oviposition, resulting in fewer eggs laid (approximately
10% from their capacity) in the container, with a lower hatchingrate (41%), and a very low proportion of larvae reaching adulthood(<1%). Although the laboratory results for An. dirus were excellent, its application in the field may be limited by the difficulty of
attaching HATS in breeding sites, such as river streams. However,
HATS has numerous advantages. First, it is safe to humans as particle sizes >100 nm cannot penetrate human skin (Schneider et al.
2009). Second, it can affect all stages of mosquitoes owing to its
ovicidal, larvicidal, and repellent effects, as well as submersion ofgravid females when laying eggs. Third, it has no smell, unlike commonly used chemicals in vector control programs, such as temephos,
which has a strong smell, and thus most people dislike using it(Phuanukoonnon et al. 2006). Thus, future studies should formulatehydroxyapatite-binding silver/titanium dioxide particles in the formof granules or tablets, which are easy to apply in the container. The
effective dose, longevity, and resistance of mosquitoes against thecompound require further investigation.

Acknowledgments
We thank the staff of the Insecticide Research Unit, Departmentof Medical Entomology, Faculty of Tropical Medicine, Mahidol
University for their assistance.

Funding
This research was supported by the collaborative research funds (FY
2018-2022) of DR.C Medical Medicine Co., Ltd., Tokyo, Japan.

Conflicts of Interest
N.I. and D.Y. are employees of DR.C Medical Medicine Co., Ltd.,and N.O. is the CEO. All other authors declare that they have noconflicts of interest.

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