Oviposition and Embryotoxicity of Indigofera suffruticosa on Early Development of Aedes aegypti (Diptera: Culicidae)

Aqueous extract of Indigofera suffruticosa leaves obtained by infusion was used to evaluate the oviposition, its effect on development of eggs and larvae, and morphological changes in larvae of Aedes aegypti. The bioassays were carried out with aqueous extract in different concentrations on eggs, larvae, and female mosquitoes, and the morphological changes were observed in midgut of larvae. The extract showed repellent activity on A. aegypti mosquitoes, reducing significantly the egg laying by females with control substrate (343 (185–406)) compared with the treated substrate (88 (13–210)). No eclosion of A. aegypti eggs at different concentrations studied was observed. The controleclodedin 35%. At concentration of 250 μg/mL, 93.3% of larvae remained in the second instar of development and at concentrations of 500, 750, and 1000 μg/mL the inhibitory effect was lower with percentages of 20%, 53.3%, and 46.6%, respectively. Morphological changes like disruption on the peritrophic envelope (PE), discontinued underlying epithelium, increased gut lumen, and segments with hypertrophic aspects were observed in anterior region of medium midgut of larvae of A. aegypti. The results showed repellent activity, specific embryotoxicity, and general growth retardation in A. aegypti by medium containing aqueous extract of I. suffruticosa leaves.


Introduction
The mosquito Aedes aegypti Linnaeus is a vector and promotes the spreading of four serotypes of dengue virus. However, a decrease in the effective vector control has been described due to larval tolerance to chemical insecticides [1]. The incidence of classical and hemorrhagic dengue fever in 2007 registered by the Brazilian Federal Organ was 559 954 cases, with 158 deaths in the country [2]. Despite significant advances in the techniques used for its control during recent decades, the mosquito A. aegypti continues to pose serious public health problems [3]. A dengue vaccine is still under development, and vector control is the only practical measure towards the reduction of dengue disease [1].
It has been demonstrated that insect gut is the target of many insecticidal compounds. Transmission electron microscopy of A. aegypti larvae treated with an aqueous extract of Derris urucu showed histological alterations in the midgut, and larval mortality was associated with peritrophic matrix damage [4]. The peritrophic matrix of insects is constituted by proteins, glycoproteins, proteoglycans, and chitin, and its integrity is important for digestive processes as well as for protection against invasion by microorganisms and parasites [5]. Plants have been evaluated as sources of natural insecticides against A. aegypti, and larvicidal bioassays have been conducted using third (L3) and fourth (L4) instars or comparing the effect of plant extracts on larval development of L1-L4 [6]. Various studies have addressed the possibility 2 Evidence-Based Complementary and Alternative Medicine of using the embryo culture technique as an assay for embryotoxic potential of xenobiotic compounds [7].
Indigofera suffruticosa Mill (Fabaceae) is a plant found in tropical and subtropical areas and well adapted to growth in semiarid regions and soil of low fertility [8]. This plant occurs in Brazil Northeast countryside and has intensive popular use in the treatment of bacterial and fungi infections, inflammations, and other diseases such as epilepsy in human and animal models [9,10]. In Brazil, the plants have been used as an infusion or decoct (flavor extract by boiling 1 L of hot water/5 g of leaves) [9].
A chemical investigation of this species (I. suffruticosa) in Natural Products Alert (NAPRALERT) [11] and Chemical Abstracts databases has revealed the presence of alkaloids, flavanoids, steroids, proteins, carbohydrates, and indigo.
In the present study, we have investigated the process of oviposition, early development on eggs and larvae of A. aegypti, and morphological changes in larvae treated with aqueous extract from leaves of I. suffruticosa. Adult mosquitoes (F0 generation) were fed with 10% glucose and with chicken blood and were reared in a room maintained at 27 • C in humidified cages. Eggs of these mosquitoes were counted using a stereoscopic microscope. The larvae generated were fed with commercial cat food. Eggs and the 1st instars larvae were used in the experiments.

Preparation of the Extracts.
Leaves (75 g) were weighed and chopped. The plant material was successively extracted in infusion with solvents of increasing polarity (hexane, ethyl acetate, and methanol). The solvents were removed by rotary evaporation. The percentage yields were hexane (0.67%), ethyl acetate (0.39%) methanol (3.9%), and (w/w) in terms of newly collected plant material. After the extraction processes with the aforementioned solvents, the same plant material was extracted with distilled water, resulting in the aqueous extract. To the egg-laying evaluation, 25 mL of aqueous extract was used with female mosquitoes. The other part of extract was lyophilized, and the dried powder plant material (4.2%) was stored at 20 • C. This dry residue aqueous extract was homogenized using 100 µL of distilled water in microcentrifuge tubes, then diluting in water to the appropriate concentration 250, 500, 750, and 1000 µg/mL to evaluate the embryotoxicity on eggs and larvae.

Oviposition Bioassay.
During 4 consecutive days, 90 female mosquitoes of A. aegypti were stored in polypropylene cages (30 × 30 × 30 cm) (Bugdorm-I, Mega View Science Education Services, Taiwan) with sacarose solution 10% at 25 • C. Females were exposed to 18 substrates (paper filter) with distilled water (9 substrates) and 25 mL at 30% of aqueous extract of I. suffruticosa (9 substrates). The quantification of the eggs was assessed by observation under a stereomicroscope (1.2x). The oviposition bioassay was assayed as recommended by the World Health Organization [15].

Embryotoxicity
Bioassay. Aedes aegypti L., whose common name is dengue mosquito, belongs to the Arthropoda Phylum, Hexapoda Class, Diptera Order, and Culicidae Family. The effect of aqueous extract of I. suffruticosa leaves on egg outbreak and larval development of A. aegypti was assayed as recommended by the World Health Organization [15]. Eggs and larvae of A. aegypti were exposed to the extract in concentrations of 250, 500, 750, and 1000 µg/mL. Preliminary bioassay was performed using 40 eggs that were hatched in mineral water (200 mL) at 26 • C-28 • C. The test using larvae (n = 15, 1st instar) were carried out in duplicate for each concentration. Larvae were placed into 200 mL disposable plastic cups containing 25 mL of the test solution and incubated at 27 • C. The developmental stages of larvae was determined at the start of the experiment (0 h) and 24, 48, and 72 h thereafter, and developmental stages were assessed by observation under a stereomicroscope (1.2x).

Morphologic Study of A. aegypti Larvae. Mosquito (A.
aegypti) larvae from control and treated groups were fixed with formaldehyde (2.5%) for morphologic evaluation and were photographed using a digital video camera (Leica) connected to an inverted microscope (magnification of 200x.).

Statistical Analysis.
We used Mann-Whitney (P < 0.001) using the SigmaStat (3.5 version) between the control and tested groups. The oviposition results were expressed in media (min-max).

Embryotoxicity Bioassay.
No eclosion of A. aegypti eggs in the different concentrations studied was observed. The same number of eggs (n = 40) was used as a control that ecloded in 35% (Table 1).    The embryonic development of larvae of first instar (L1) of Aedes aegypti was observed from 0 to 72 h using concentrations from 250 to 1000 µg/mL of aqueous extract of I. suffruticosa leaves. Table 2 compares the effect of extract of I. suffruticosa at different concentrations.

Morphologic Study of A. aegypti Larvae.
Control Live L2 on distilled water (Figure 2(a)) and treated live L2 on aqueous extract of I. suffruticosa (Figure 2(b)) after 72 h of incubation were evaluated using inverted optical microscope.
Morphological observation of anterior region of medium midgut of larvae of Aedes aegypti in early development treated with aqueous extract of I. suffruticosa showed disruption on the peritrophic envelope (PE) structure consequently resulting in a discontinued underlying epithelium, increased gut lumen, and segments with hypertrophic aspects in comparison with control larvae. The developmental delay is directly dependent of morphological changes that occur when the larvae are growing in contact with different substances of the extract.

Discussion
The purpose of this study was to determine the repellent and toxic effects of Indigofera suffruticosa on oviposition and embryonic development of Aedes aegypti.
The results showed significant repellent effect on egg posture and specific embryotoxicity and general growth retardation on A. aegypti by medium containing aqueous extract of I. suffruticosa leaves.
Studies reporting repellent effect with Indigofera species were not found in literature, but many plants from the family Lamiaceae are toxic for insects including Ocimum basilicum, O. gratissimum, O. americanum, Cymbopogom nardus, Alpinia galanga, Syzyaium aromaticum e Thymus vulgaris, Mentha, Eucalyptus maculata citriodon, and Tagetus e Lantana camara, and they have been studied as natural alternative repellents [16].
A. aegypti eggs did not outbreak and larvae in early development showed an increase of abnormalities, mainly in the peritrophic envelops at different concentrations. At 250 µg/mL concentration the extract could affect one of the phases of the life cycle of A. aegypti. Higher incidences of specific embryotoxicity were found at concentrations that also caused general growth retardation [15]. The in vitro counterpart of teratogenicity was defined as specific embryotoxicity that could be distinguished from general retardation of growth and development of the embryo. By using this definition, general toxic effects are not considered to indicate specific embryotoxicity, since general toxicity will be induced by virtually any compound if added at sufficiently high concentrations [15]. Four compounds tested that were not teratogenic in vivo: amaranth [17] and isoniazid [18] had only growth retarding and/or lethal effects at high concentrations in vitro, whereas penicillin [19] and saccharin [20] did not show any effect at the highest concentration tested in culture. However, the most important confounding factor in the use of whole embryo culture as a screening test is likely to be the experimenter's judgment regarding the scoring of specific embryotoxicity, especially the distinction between specific toxicity, on the one hand, and general toxicity and growth retardation on the other hand. The interpretation of malformed and retarded embryos is complicated further when effects occur at low incidences, as described in the present study for extract of I. suffruticosa. Aqueous extract of I. suffruticosa leaves was studied for adverse effects in preimplantation mouse embryos. Two-cell mouse embryos were cultured for 94 h in human tubal fluid medium (HTF),  and the extract at a concentration of 5 mg/mL showed a development from morula to blastocyst stages similar to the controls, and at a higher concentration (10 mg/mL), all embryos persisted at the two-cell stage [12].
In vertebrates, mucus is the primary secreted layer, lining and protecting the intestinal epithelium, while assisting the digestion process [21]. However, insects do not possess a typical mucus layer in the digestive tract, and instead, their midgut is lined by a unique protective structure, the peritrophic envelop (PE) [22]. The PE is a mucinous structure, which is uniquely different from vertebrate mucus by its incorporation of chitin, resulting in proteinaceous structure reinforced by chitin fibrils [23]. Despite these important functions, the biochemical properties and molecular biology of PE formation is still poorly understood [23].
This experimental study demonstrated that extract could act promoting morphological changes on PE in larvae of A. aegypti. Furthermore, the inhibition of PE formation severely affected the early development of larvae. In controlling second instar larvae of A. aegypti, the anterior region of medium midgut was recovered by a continued PE. However, morphological observation of larvae submitted to aqueous extract of I. suffruticosa leaves showed disruption on the PE structure. Clearly, we are far away from completely elucidating the mechanisms of I. suffruticosa to induce growth retardation in animal models. However, studies from our group also demonstrated that this plant is an extremely powerful inducer of cancer cell death and possibly the bioactive compound from I. suffruticosa could act binding many molecular targets inside the cell activating alternative apoptotic pathways or inducing mitotic catastrophe which indicates a form of cell death that is caused by aberrant mitosis by caspase 3 activation and oligonucleosomal DNA degradation [24]. On the whole, all the aforementioned data indicate that I. suffruticosa can induce cell death via different molecular pathways and with different executing mechanisms, that is classical apoptosis, but also mitotic catastrophe. These activities and the main recognized molecular targets of I. suffruticosa are depicted in Figure 3. Due to these actions, I. suffruticosa can impinge upon different conditions (represented as circles in the Figure 3).
Plants and their derivatives were used for controlling and eradicating mosquitoes and other domestic pests before the advent of synthetic organic chemical [21].
Evidence-Based Complementary and Alternative Medicine 5 The use of plant extracts in insects control is an alternative pest control method for minimizing the noxious effects of some pesticide compounds on wildlife, livestock, non target insect species, and the environment [25].
There is a general lack of effective and inexpensive chemotherapeutic agents for treating this disease that occurs in the developing world. In addition, specimens from sites where there has already been intensive use of the larvicide in dengue control programs are more likely to show resistance to the larvicide, and it has become a severe problem [26].
In this sense, new insecticides of herbal origin discovered through ethnopharmacological studies have shown interesting results. Our laboratory has initiated and developed original investigations, and we have evaluated the embryotoxicity caused by compounds from natural extracts of plants.
Purification of the bioactive component(s) from Indigofera suffruticosa is underway, and further investigations may improve our understanding of possible developmental changes from aqueous extract of this plant used in folk medicine.