Ascogregarina Taiwanensis Interfere in the Performance of Aedes Albopictus and in the Susceptibility of Aedes Aegypti to Temephos and Azadirachta Indica Oil

Background: Aedes albopictus and Aedes aegypti are mosquitoes commonly adapted to tropical and subtropical regions. These vectors can transmit different types of arboviruses causing a serious concern to public health. New alternatives for the vector/arboviruses control are emerging, and in this sense the protozoan Ascogregarina taiwanensis may present potential as a biological control agent against these mosquitoes. Methods: To evaluate the effects of protozoan A. taiwanensis, mosquitoes were parasitized with a solution containing oocysts and evaluated to lifetime, fertility, fecundity for Ae. albopictus and for Ae. aegypti interaction with Azadirachta indica and Temephos. Results: In this work it was possible to observe the protozoan morphology in mosquitoes Ae. albopictus, as well its negative inuence on mortality, 73% and non-parasitized was 44%. The number of eggs oviposited by parasitized females of Ae. albopictus was lower (3,490) than for the non-parasitized females (5,586). In addition, the hatchability and/or viability of these eggs were also lower for the parasitized females (63%) than the non-parasitized ones (74%). For Ae. aegypti mosquitoes, a synergism between the use of A. taiwanensis associated with a chemical insecticide and a botanical insecticide was observed. The results demonstrate that when Ae. aegypti larvae was parasitized by A. taiwanensis and exposed to the oil of Az. indica or to the organophosphate Temephos present a greater mortality. Conclusion: It was notable that A. taiwanensis can be a potential for biological control and adjuvant of insecticides. We also provide important information about the maintenance of A. taiwanensis in laboratory.


Background
Every year, around 17% of all infection diseases are caused by vector-borne diseases [1]. Among these vectors, the mosquitoes have playing the main role in the transmission of several arboviruses like dengue, Zika and chikungunya [2][3][4][5][6].
Aedes aegypti and Aedes albopictus are worldwide distributed [7] and have highly anthropophilic and opportunistic behavior [8,9]. As competent vectors for several human arboviruses, these mosquito species are responsible for major public health concern.
Different methods for mosquito control have been suggested, and these methods can be classi ed as biological, genetic, environmental, mechanical and chemical [10]. Meanwhile, due to the problems surrounding arboviruses in recent years and the resistant selection of some mosquito populations through continuous insecticides use [11,12], alternative methods for vector control must be thought.
Studies have shown that the synergism between microorganisms and chemical insecticides can be useful when comparing to the exclusive insecticide use [13,14]. In addition, several other methods of control including with microorganisms have been proposed in the last years, such as: growth regulators, chitin synthesis inhibitors and behavior modi ers that can be in uenced by virus, bacteria, fungi and protozoa [15][16][17][18][19][20][21][22][23].
Gregarines are protozoan that can naturally parasite a huge variety of insects [24]. Among these insects, some species of mosquito can harbor some gregarines bellowing to the genus Ascogregarina (Eugregarinida: Lecudinidae) [25]. In this way, [26] have proposed that gregarine parasite can interfere negatively in their biological host development and this in uence depends on their environmental distribution.
Studies using A. taiwanensis as biological control and its in uence in biological development of mosquitoes must be better understood, besides that the knowledge of laboratory maintenance of this protozoa is poorly known. So, after the encounter of Ae. albopictus and Ae. aegypti harbouring A. taiwanensis in south Brazil [31], we established this protozoan in laboratory conditions. So, we could evaluate the in uence of this gregarine on some biological aspects of Ae. albopictus. Besides that, we induced their parasitism in Ae. aegypti in order to evaluated it susceptibility to insecticides after being infected. Such study could provide new information about the parasitism of A. taiwanensis in Aedes mosquitoes contributing for the studies in control of these vectors.

Mosquito strains
Two mosquito strains were used in this study: Ae. aegypti (Rockefeller) and a non-parasitized Ae. albopictus collected in the eld. Larvae were fed using pet food (Purina® Cat Chow®) 200 mg/mL, three times a week. Adult mosquitoes were reared under a 14 h light/10 h dark photoperiod, at 25°C in an incubator (132FC ELETROlab®). Honey solution (10% w/v) was continuously provided to adult males and females, while females were blood-fed on mice Mus musculus (Ethic Committee of Animals − 19843), twice a week in order to obtain eggs for the bioassays and colony development.

Ascogregarina taiwanensis reared in laboratory
Aedes albopictus larvae naturally harboring A. taiwanensis (GenBank, NCBC KM387708) were collected from traps (plastic pots and tires) in Tubarão/SC -Brazil in the year of 2014 and brought to the laboratory (Prophiro et al. 2017). The emerged adults were kept under controlled conditions as described below. After blood feeding, an arti cial breading place containing 500 mL of water was offered to female for oviposition, and consequently where oocysts could be released, and posteriorly parasite new healthy larvae. To become parasitized, these larvae were separated in two groups: one group with all larval stages together and one group with larvae separated by stage. The con rmation of A. taiwanensis infection in the new generations of mosquitoes was carried out based on morphology of parasites, according to Prophiro (2017). This new generation of Ae. albopictus harboring A. taiwanensis was used to infect a laboratory reared Ae. aegypti and a eld collected Ae. albopictus in order to conduct the bioassays described below. This work was registered by Brazilian Genetic System SISGEN (A11AEC2).

Maintenance of Ascogregarina taiwanensis in laboratory
As cited above the oocysts of A. taiwanensis used for the infection of Aedes mosquitoes came from an adult colony of Ae. albopictus collected in the eld naturally parasitized. A solution containing oocysts was produced using a similar method described in Beier and Craig (1985), where 100 parasitized adults were homogenized in 100 mL of ltered water, and posteriorly diluted in 3 liters of water containing Ae. albopictus and/or Ae. aegypti larvae to infect such populations. After 24 hours of infection, the larvae were transferred to other container containing ltered water, in order to avoid reinfection in different days.

Morphology of Ascogregarina taiwanensis in different stages of vector Ae. albopictus
After infection, a sample of 50 larvae (2nd, 3 rd, and 4th instars), 50 pupae and 50 adults (25 males and 25 females) of Ae. albopictus obtained through arti cial transmission was dissected to con rm the presence of the protozoa and photographed in Microscopic photographs (OLYMPUS CX31-P and ZEISS STEMI 200 C).
In uence of Ascogregarina taiwanensis on the performance of Aedes albopictus After obtaining the Ae. albopictus population harboring A. taiwanensis (15 females and 15 males) were separated in three cages (30 x 30 x 30 cm). The same was made for control group (without A. taiwanensis), totaling six cages with 180 mosquitoes. Each group was treated with 10% honey solution.
After oviposition of females the eggs were counted and conditioned in a climatized room. Three weeks after oviposition, the eggs were placed in plastic trays with water and food for stimulating larvae hatching. The development through larvae to the adult stage were monitored daily and the longevity of these mosquitoes was monitored every 48h. This experiment was carried out three times at different days.

Susceptibility of Aedes aegypti to insecticides when parasitized with Ascogregarina taiwanensis
In these bioassay two insecticides were used: Temephos technical grade 96% lot #SZBD128XV manufactured by the laboratory "Fluka Analytical", St. Louis, MO 63103 -USA, and Azadiracta indica (Neem oil) lot 44796-04 manufactured by the laboratory "Handa Fine Chemicals", West Sussex -USA.
Both insecticides were calibrated with Ae. aegypti Rockefeller strain.
For the bioassays third instar late and early fourth instar larvae of Ae. aegypti were used in two groups: non-parasitized (control group) and parasitized with A. taiwanensis. Three replicates of 15 larvae, totaling 45 larvae/concentration + 15 control larvae were exposed to six different concentrations of Temephos (0.009-0.024 ppm) or Az. indica oil (14-169 ppm) in 100 mL of solution. A total of 90 larvae for each product and population were exposed to solvent ethanol and Tween 80 (polysorbate) as control. Larval mortality was veri ed after 24h of exposure. Moribund larvae and unable to reach the surface of the water when touched with a needle were considered dead (WHO, 1981). The surviving larvae were discarded, and the bioassays were reproduced three times on different days for each product.

Statistical analysis
Kruskal-Wallis non-parametric test (KW) was used to detect differences in the treatments in relation to the different generations and strains. When the differences were detected, the Multiple Comparisons test was applied through the STATISTICA 7.0 program, with signi cance level P < 0.05. The Probit GW-Basic program was used to determine lethal concentrations. Two-way ANOVA of GraphPad Prism 5.03 was used to analyze the results, with a signi cance level of 5%. A t test was used to compared differences between oviposition (parasitized and non-parasitized) and in viability of these eggs.

Aedes albopictus infection by Ascogregarina taiwanensis
The morphology of trophozoites usually had appearance of comma or was rounded. The average size of this protozoan was: second instar (58.5 µm), third instar (77 µm) and the fourth instar (168.1 µm). The location of the trophozoites was normally observed at the end of midgut, next to the Malpighian tubule. The parasites could be observed in second instar larvae ( Fig. 1A and B), third instar ( Fig. 1C and D), fourth instar ( Fig. 1E and F), and pupal stage ( Fig. 1G and H). The presence of the gametocytes in the adults was also observed (Fig. 1I). Due to the small size of the rst instar larvae of Ae. albopictus, only in 2nd, 3 rd, and 4th instars A. taiwanensis could be observed.
When larvae of the second group were separated by stage and exposed to infection by oocysts resulting from the macerate of parasitized adult of Ae. albopictus, the second and third instar demonstrated greater potentiality of being parasitized, showing 100% of infection. The fourth instar larvae did not show any trophozoite in their digestive system. May be that this larva stage does not provide a viable time to the development of oocysts into trophozoites, because in a short time (about 48 hours) turns into pupa.
In uence of Ascogregarina taiwanensis on the performance of Aedes albopictus The population of Ae. albopictus parasitized by A. taiwanensis showed shorter period of longevity when compared the non-parasitized population (Fig. 2). Signi cant differences were observed in mortality among the parasitized and non-parasitized population (independent of sex) (KW = 12.25, gl = 1, P < 0.05, X 2 = 6.13, P = 0.0005). No signi cant differences were observed in mortality over the days analyzed, both in the parasitized and non-parasitized populations (P = 0.41 and P = 0.47, respectively).
The number of eggs oviposited by parasitized females of Ae. albopictus was lower than for the nonparasitized females, however there is no signi cant differences between them (Table 1). In addition, the hatchability and/or viability of these eggs were also lower for the parasitized females than the nonparasitized ones (Table 1). Signi cant differences were observed in egg viability of the parasitized and non-parasitized populations (p = 0.0143). These results are like those obtained by Comiskey et al. (1999), were Ae. albopictus parasitized by Ascogregarina sp., presented a decrease in the reproductive capacity of females, even with high nutrient conditions.

Susceptibility of Aedes aegypti to insecticides when parasitized with Ascogregarina taiwanensis
In our bioassays there was higher larval mortality of Ae. aegypti after exposure to Az. indica oil, when this vector was parasitized by A. taiwanensis (Fig. 3A). In the presence of the parasite, the LC 50 was 0.815 mg/L whereas in the non-parasitized group the LC 50 was 1,812 mg/L. The results showed that there was a signi cant difference between the values of mortality comparing the parasitized and non-parasitized group (P < 0.001). There was no mortality in the control groups (polysorbate and water).
For Temephos treatment, a higher mortality was also observed where there was synergism between the protozoan A. taiwanensis and Temephos (Fig. 3B). In the presence of the parasite the LC 50 was 0.025 mg/L whereas without the parasite the LC 50 was 0.063 mg/L. The results showed that there was a signi cant difference between the values of mortality comparing the parasitized and non-parasitized group (P < 0.001). There was no mortality in the control groups (ethanol and water).

Discussion
In this work we demonstrate that when Ae. aegypti larvae was parasitized by A. taiwanensis and exposed to the oil of Az. indica or to the organophosphate Temephos induce a higher mortality. Mosquitos' mortality (parasitized males and females) was 73%, while mortality of the non-parasitized was 44%.
These results are like those reported by [32,33], which obtained reduction in the longevity for Ae. aegypti parasitized by A. culicis and Ochlerotatus triseriatus parasitized by Ascogregarina barretti. These authors also observed prolongation of the larval stage and reduction of adult size for both species of mosquitoes [32,33]. [34], observed that Ae. albopictus parasitized by Ascogregarina sp. presented higher mortality of immature stages when larvae were under nutrient. These morphological observations in all protozoa stages were like that found by Lien and Levin (1980).
These results are like those obtained by Comiskey et al. (1999), were Ae. albopictus parasitized by Ascogregarina sp., presented a decrease in the reproductive capacity of females, even with high nutrient conditions. The synergism between microorganisms and insecticides inducing higher mortality was also reported by [14]. This author veri ed that when larvae of Ae. aegypti were exposed to Az. indica and the fungus Metarhizium anisopliae (5×10 5 conidia/mL) presented higher mortality. Similarly, [13] observed that when Bacillus thuringiensis var. israelensis is used with Temephos in Ae. aegypti, a 90% greater larval mortality is obtained in the rst hour of exposure, compared to the group treated with Temephos alone.
The compounds of Az. indica have several forms of action, which may act in an antiparasitic, antihelmintic, antimicrobial and other forms [35,36]. In the present work, the higher mortality of parasitized Ae. aegypti when exposed to Az. indica may be related to antiparasitic action. According to [37], extreme variations of physiological conditions in association with parasitic infection can cause necrosis in the cells, resulting in direct damage to the plasma membranes of the host. Thus, we can suggest that if there was an antiparasitic action of Az. indica on the gregarine facilitated the insecticidal activity of this oil on the larvae.
Although the A. indica concentration is higher than Temephos in the dosage values, it is noteworthy that there are reports that the survival of Ae. aegypti exposed to more than 0.02 mg/L of Temephos indicates the possibility of resistance among the population tested (Brown, 1986;Denham et al. 2015;Arslan et al. 2015).
According to [38] new methods for Aedes vector control aimed at reducing the use of chemical insecticides should be urgently prioritized. Thus, we believe that integrated and interleaved control may also reduce the pressure on the selection of individuals who are resistant to routinely used chemical insecticides. The results obtained, indicate that A. taiwanensis negatively in uences its host, in this case both Ae. albopictus as Ae. aegypti. In this way, we believe that this gregarine has potential for biological control of vectors.
The synergism between microorganisms and insecticides inducing higher mortality was also reported by [14]. This author veri ed that when larvae of Ae. aegypti were exposed to Az. indica and the fungus Metarhizium anisopliae (5×10 5 conidia/mL) presented higher mortality. Similarly, Andrande and Modolo (1991) observed that when Bacillus thuringiensis var. israelensis is used with Temephos in Ae. aegypti, a 90% greater larval mortality is obtained in the rst hour of exposure, compared to the group treated with Temephos alone. Interestingly, it has also been reported that Ae. albopictus infected with Ascogregarina reduces its competitiveness in the habitat with different larvae such as Ae. triseriatus [39]. In addition, it has already been shown that the propitious infection by Ascogregarinas can impact the Ae. Albopicuts microbiota. [40].
The compounds of Az. indica have several forms of action, which may act in an antiparasitic, antihelmintic, antimicrobial and other forms [35,36]. In the present work, the higher mortality of parasitized Ae. aegypti when exposed to Az. indica may be related to antiparasitic action. According to Golstein and Kroemer (2007), extreme variations of physiological conditions in association with parasitic infection can cause necrosis in the cells, resulting in direct damage to the plasma membranes of the host. Thus, we can suggest that if there was an antiparasitic action of Az. indica on the gregarine facilitated the insecticidal activity of this oil on the larvae. Although the A. indica concentration is higher than Temephos in the dosage values, it is noteworthy that there are reports that the survival of Ae. aegypti exposed to more than 0.02 mg/L of Temephos indicates the possibility of resistance among the population tested [41][42][43] According to Guirado and Bicudo, (2009) new methods for Aedes vector control aimed at reducing the use of chemical insecticides should be urgently prioritized. Thus, we believe that integrated and interleaved control may also reduce the pressure on the selection of individuals who are resistant to routinely used chemical insecticides. The results obtained, indicate that A. taiwanensis negatively in uences its host, in this case both Ae. albopictus as Ae. aegypti. In this way, we believe that this gregarine has potential for biological control of vectors.

Conclusions
In this work we demonstrate the parasitism capacity of Ascogregarina taiwanensis in Aedes albopictus and show its impact on the different stages of development of the mosquito, we show the decrease in its longevity, quantity of eggs and hatching. We also show that larvae of Aedes aegypti parsitated with the protozoan, have a synergistic effect with Temephos and Azadiracta indica oil, increasing mortality and decreasing their lethal concentration. We believe that this is another indication of the use of new biological agents for vector control and that