Exploring the Insecticide and Acaricide Potential of Development Regulators obtained from Restinga vegetation from Brazil

Abstract

Abstract: As a part of our continuing search for insect and arthropod development regulators from Brazilian restinga vegetation of the Rio de Janeiro State, crude extracts, purified fractions and essential oils were submitted to screening tests seeking for biological activities on the development of the insects Rhodnius prolixus, Dysdercus peruvianus, Oncopeltus fasciatus and Rhipicephalus (Boophilus) microplus. Up to now, 102 secondary metabolites have been detected in the fractions, among them monoterpenes, sesquiterpenes and two triterpenes which were obtained from the species, Eugenia sulcata, Pilocarpus spicatus, Manilkara subsericea, Myrciaria floribunda and Zanthoxylum caribaeum. These secondary plant metabolites are considered of interest for the use of studies related to arthropod endocrinology, vector-parasite interaction system, and population control of vector insect and agricultural pest. The observed biological activities were surprisingly high, involving increased mortality, molting and metamorphosis inhibition, paralysis, corporeal deformities, apparition of permanent nymphs, of adultoids and juvenoids, partial or total inhibition of oviposition and egg hatching. These compounds are now being studied further to determine if they may or may not be useful in controlling insect populations and/or interfere with the life cycle and vector transmission of parasites to animal and human populations.

Key words
arthropods; essential oil; green pesticide; insect pests; insect vectors; insect growth regulators

INTRODUCTION

Restinga vegetation is a mosaic of plant communities growing at sandy coastal plains found along much of the tropical, subtropical and temperate Brazilian coast (Araujo and Henriques 1984, Araujo 1992).

They are formed by marine, fluvio-marine or wind sediments originated from the Quaternary Period. (Marques and Oliveira 2004, Kelecom et al. 2002a). Such environments display many geomorphological formations, invariably with low availability of resources, mainly due to the low capacity of retention of water and nutrients in sandy soils. (Rosado and De Mattos 2007). The estimative for the Brazilian restinga vegetation is more than 4,000 species, only to Rio de Janeiro state, 1,005 species have already been registered (Sá 2014, Araujo 1992).

According to the Chico Mendes Institute for Biodiversity Conservation (ICMBio, 2017), the Restinga de Jurubatiba National Park, created on April 29, 1988, is a federal conservation unit with 14,922.39 hectares, 44 km of coast and 18 coastal lagoons. It is located between latitudes 22º and 22º23’S and longitudes 41º15 ‘and 41º45’W (Fig. 1) incorporating the cities of Macaé, Carapebus, and Quissamã in the state of Rio de Janeiro (Santos et al. 2004). According to Araújo (2000), the Restinga of Jurubatiba possesses a great diversity of vegetal species, almost completely unknown from the chemical and pharmacological points of view. It is thus reasonable to expect that new metabolites with very uncommon structures may be encountered, and that a number of them may be endowed of biological activities. Hence, due to the biological potential of secondary metabolites, plants of such a peculiar biome were investigated looking for insecticide and acaricide potential, aiming agriculture pests control.

Figure 1
Map showing the coordinates of Restinga de Jurubatiba National Park.

The agricultural sector is nowadays one of the main sources of income and productivity in the world economy. According to the Food and Agriculture Organization of the United Nations (FAO 2013), about 75% of the world agricultural economy is generated in developing countries, and in many of these this sector contributes 30% of gross domestic product (GDP).

In Brazil, agrobusiness plays a major role in the economic scenario, being a fundamental component for economic performance and integration (Campanhola, 2005). In the year 2013, Brazilian agrobusiness, from the agricultural raw material to its industrialization and commercialization, including the sectors that supply feedstock, machinery, and implements, accounted for 22.5% of the GDP, that is about USD 300.000.000.000 (Barros et al. 2014). According to the literature, the increase in agricultural production in Brazil is directly related to technological development, which is derived from studies and directed research (Campanhola, 2005).

The presence of populations of the phylum Arthropoda, mainly of the class Insecta and order Ixodida that act as agricultural plagues or parasites of animals are considered as a serious economic problem since the annual losses of productivity of crops and animal breeding reach of significant proportions. The use of chemicals to control populations of insects and ticks to improve economic investment may result in toxicological impacts on animals, man and the environment, and are often more damaging than the pests intended to be controlled (Faria et al. 2007). Among the main damages observed are: (i) increased resistance by pests to current insecticides due to changes in the rate of penetration and inactivation of the biochemical mechanisms of these insecticides, (ii) the emergence of secondary pests as a result of the elimination of natural enemies, and (iii) the elimination of non-target organisms, e.g. insects useful for pollination. However, because of their proven effectiveness and rapid action, conventional pesticides (insecticides) are still the most important tool for population control programs of disease vectors, mites and agricultural pests, even though the arsenal of effective products has been decreasing due to the development of resistance to the most commonly used insecticides (Zaim and Guillet 2002).

Resistance development is a skill acquired by an organism’s lineage in tolerating doses of a toxic product that is lethal to most of the normal population of the same species. Ticks and insects have a rapid life cycle and accelerated reproduction, where one female breeds hundreds to thousands of individuals. This favors the emergence of populations with new genetic characteristics. The spread of insecticide resistance in insect populations is related to the frequency of their use and results not only from the selective pressure of these toxic compounds on these populations but also from the inherited characteristics of the insect species involved (Hemingway and Ranson 2000).

Individuals with advantageous mutations related to the resistance phenotype are more likely to survive insecticide treatments and contribute to offspring than susceptible individuals, resulting in increased frequency of the gene conferring resistance in the next generations (Beaty and Marquard 1996).

Nowadays, liquid solutions and sprays containing organochlorines, organo-phosphates and synthetic pyrethroids are commonly used in the control of arthropods important for public health and management of modern agriculture (Mello et al. 2007).

In the specific case of acaricides, such products are classified as “systemic” or “by contact”. The formers are applied by injections into the bovine host, while the latter’s can be applied by spraying or immersion of the cattle or by the pour on method (application on the animal’s back) (Rajput et al. 2006).

Since the use of insecticides and acaricides of first generation (inorganic compounds) and second generation (organochlorines, organophosphates, carbamates and pyrethroids) for the chemical control of agricultural pests and insect vectors is banned in many countries throughout the World, a public demand for safe technologies became a major concern, and this implies in the development of products with more selective target sites and low risk for non-target organisms and the environment (Graf 1993, Faria 2009). Integrated management of agricultural and arthropod pests of medical and veterinary interest should now be considered, consisting of the use of controlled techniques capable of increasing efficiency in combating these organisms, simultaneously improving the possible use of economic investment and avoiding harmful effects on the environment (Kogan 1998, Smith and Raupp 1986).

The European Union (EU) regulatory system is the strictest in the world and Pesticides in the EU are constantly monitored. Only substances for which there is objective evidence of safe use are approved. (European Commission access in 17 04 2018). Organochlorine and organophosphates pesticides are synthetic pesticides widely used all over the world, which have vast application in the chemical industry and in agriculture. Residues of pesticides can be found in a great variety of everyday foods and beverages. These compounds are known for their high toxicity, slow degradation and bioaccumulation. (Jayaraj et al. 2016).

Alternative strategies for the control of arthropod, tick and insect populations are available, for example the use of fungi, BT toxin, nematodes etc. Secondary metabolites participate in physiological processes essential to plants. They are also responsible for the relationship of plants with the environment in which they interact, defending against microorganisms, herbivores or ultraviolet rays, attracting pollinating agents, or even assisting in symbiotic relationships with other beings. They are also responsible for several pharmacological activities that the plants present. Their production is modulated according to the needs of the plant itself, which is synthesized and degraded (Taiz and Zeiger 1991).

Studies on insect physiology and plant chemistry have revealed defensive plant strategies against insects that do not use the intrinsic toxicity of the secondary metabolites produced (Rattan 2010). These strategies promote a direct interruption in the specific physiological mechanisms necessary for the insect to successfully perform its metamorphosis, reproduction, diapause and behavior through interference with its endocrine system, regulating these and other important vital functions, thus representing a vulnerable point in the life cycle of these insects and ticks. Borges et al (2003) showed that extracts of Melia azedarach L. (Meliaceae) inhibit egg production of immersed Rhipicephalus (Boophilus) microplus ticks and Feder et al. (1988) showed azadirachtin inhibits egg production in Rhodnius prolixus. Such compounds are classified as Insect Grown Regulators (IGRs), since they interfere or are capable of inducing changes in growth and/or differentiation processes throughout the insect/ticks life cycle. These chemicals are of special interest because of their ability to act specifically on target organisms and absence or minimal biological activity for non-target organisms, in addition to the low environmental impact associated with their application, which qualifies them for use in integrated programs for the control of vectors of diseases, agricultural and livestock pests (Modal and Parween 2001).

Our investigation of IGRs has been focused on the flora of Brazilian restinga vegetation. This is a mosaic of plant communities growing at the sandy coastal plains (Magnago et al. 2011, Araujo 1992).

We here review the screening tests carried out by our group on crude extracts, purified fractions and essential oils seeking for biological activity on the development of arthropods of agricultural, veterinary or medical interest. Several fractions obtained from 5 plant genera were selected containing a number of active secondary metabolites. These metabolites are considered of interest for the use of studies related to insects and ticks endocrinology. We also discuss their scientific relevance in the current state of the art in the field of IGRs use for the population control in insect/ticks of agricultural, medical and veterinary interest.

MATERIALS AND METHODS

STUDIED MATERIAL

Native plants of the Restinga de Jurubatiba National Park, Rio de Janeiro state, Brazil were located, identified and collected by a botanist (Prof. Dr. Marcelo Guerra Santos). Voucher specimens were deposited at the Herbarium of the Faculdade de Formação de Professores da Universidade do Estado do Rio de Janeiro (RFFP). Five species will be discussed: Eugenia sulcata Spring ex Mart. (Myrtaceae), with the local name “murtinha” or “murta-preta” (Santos et al. 2009); Pilocarpus spicatus A.St.-Hil. (Rutaceae), with the local name “jaborandi-da-restinga” (Mello et al. 2007); Manilkara subsericea (Mart.) Dubard (Sapotaceae), with the local name “guracica” (Santos et al. 2009); Myrciaria floribunda (H.West ex Willd.) O.Berg (Myrtaceae), with the local name “camboim” (Tietbohl et al. 2014); and Zanthoxylum caribaeum Lam. (Rutaceae), with the local name “guando-do-mato” (Nogueira et al. 2014a, b).

Extraction purification and identification of Manilkara subsericea constituents

Fresh fruits were crushed and macerated in ethanol (EtOH) 96% (v/v) at room temperature. Filtration and concentration under vacuum afforded the ethanolic crude extract, which was then dissolved in EtOH/H₂O 90% (v/v) and partitioned with hexane to obtain, after evaporation, the hexane-soluble fraction (FH). Silica gel column chromatography (0.063-0.2 mm particle size, Vetec®) of FH. This hexane-soluble fraction (FH) was chromatographed on a silica gel column (0.063-0.2 mm particle size, Vetec®) using mixtures with increasing amounts of ethyl acetate in n-hexane. The fraction eluted at the ratio of 98:2 (v/v) n-hexane: ethyl acetate provided, after repeated crystallizations in acetonitrile, a white amorphous powder (PFT) (459.6 mg, yield 0.27%). Identification of PFT as a mixture of a and b-amyrin acetates resulted from ¹H (300MHz, CDCl₃) and ¹³C-NMR (75MHz, CDCl₃) recorded on a Varian VNMRS 300MHz spectrometer, and by GC/MS performed with a SHIMADZU GCMSQP5000 equipment (Fernandes et al 2013).

Extraction of the essential oils

Each material (E. sulcata - leaves and stems, M. floribunda - leaves, stems and flowers, P. spicatus - leaves and Z. caribaeum - leaves) was placed in a 5L distillation flask and submitted to hydrodistillation during 3-4h using a Clevenger-type apparatus. At the end, the oils were collected, dried over anhydrous sodium sulphate and stored at 4 °C until further analysis. (Rocha et al. 2012)

Chemical analysis of the essential oils

Essential oils were analyzed on a GCMS-QP5000 (SHIMADZU) gas chromatograph equipped with an electron impact mass spectrometer. The gas chromatographic (GC) conditions were as follows: injector temperature, 260°C; FID temperature, 290°C; carrier gas (helium), flow rate 1 mL/min and split injection with split ratio 1:40. Oven temperature was raised from 60°C to 290°C at a rate of 3°C/min. One microliter of each sample, dissolved in CH₂Cl₂ (1:100 mg/μL), was injected on a DB-5 column (i.d. = 0.25mm, length 30m, film thickness = 0.25μm). The mass spectrometry (MS) conditions were voltage 70 eV and scan rate 1 scan/s. The retention indices (RI) were calculated by interpolation to the retention times of a mixture of aliphatic hydrocarbons (C₉-C₃₀) analyzed in the same conditions. The identification of the substances was performed by comparison of their retention indices and mass spectra with those reported in literature. The MS fragmentation pattern of compounds was also checked with NIST mass spectra libraries. Quantitative analysis of the chemical constituents was performed by flame ionization gas chromatography (CG/FID), under same conditions of GC/MS analysis and percentages obtained by FID peak-area normalization method. (Lima et al. 2012; Mello et al. 2007, Tietbohl et al. 2012, Nogueira et al. 2014a).

Screening of biological activities

For in vivo analysis of the biological activity, the fractions, essential oils and isolated metabolites were administered to our arthropod models (see item 3.0) either orally (mixed with blood), topically (brushing the dorsal cuticle) or by continuous treatment (i.e., contact with the previously treated substrate). Periodically after the treatments, the following biological parameters were quantified: preference or refusal by untreated fertilized females to deposit their eggs on substrates treated with different concentrations of the tested compounds; viability and hatchability of the eggs deposited or relocated to these substrates; differential sensitivity of different molting stages to treatments; attraction and repellency; anatomical malformations; phagoinhibition; feed rate and excretion; weight gain and body growth; rate, time and period of moulting; death after 24 hours, and at different times of the development and longevity; oviposition (fecundity); viability and egg hatching (Mello et al. 2007, 2008, Vinturelle et al. 2017) (Fig. 2).

Figure 2
Methodological Approach and Experimental Design.

ARTHROPODS AND THEIR AGRICULTURAL, MEDICAL OR VETERINARY IMPORTANCE.

ORDER HEMIPTERA

According to Gullan and Cranston (2008), the order Hemiptera is divided into two main groups: (i) Homoptera, represented by cicadas, leafhoppers, aphids and cochonilas, and (ii) Heteroptera, consisting of bedbugs, such as Stinkbugs, water cockroaches, barbers, notonectomids and reduviids. The latter group is characterized by the presence of metapleural odoriferous glands, among which are the families Reduviidae, Pyrrhocoridae and Lygaeoidea.

RHODNIUS PROLIXUS AND CHAGAS DISEASE

More than 100 years after its discovery, Chagas disease (Chagas, 1909, 1911) is still recognized by the World Health Organization as one of the thirteen most important neglected tropical diseases in the world, is a scourge for humanity and a relevant social and economic issue in many Latin American countries. It produces important social losses in terms of mortality, absenteeism, uselessness for vital activities, and medical-social costs. Such facts are generally ignored by politicians and governments, due to poor visibility of the disease and anonymity of the population affected by the disease. Probably over 10,000 people die every year from clinical manifestations of Chagas disease, and more than 25 million people risk acquiring the disease. An estimated 8 million people are infected worldwide, mostly in Latin America. The main mechanism of transmission is through insect vector such as hematophagous Hemipterans of the subfamily Triatominae. They infect people by depositing their infected feces under the skin and mucosae after the bite. Other modalities of transmission are by transfusion, organ transplantation, orally and congenitally (WHO, 2006, OPAS, 2014). Rhodnius prolixus Sthal, 1856 (Hemiptera: Reduviidae) (Fig. 3), is an important vector of Chagas’ disease in South and Central America, traditionally used as a model for studies on insect physiology (Garcia and Azambuja 1991), strategies of vector control (Garcia and Azambuja 2004), and parasite-vector interactions (Cortez et al. 2002, Garcia and Azambuja 1991, Gonzalez et al. 2000, Kollien and Shaub 2000, Kelecom et al. 2002b, Azambuja et al. 2017). All these studies provide us with a significant amount of information about molecular factors in the neuroendocrine system and digestive tract that may be involved with fundamental stages of its life cycle (i.e., molting, reproduction) and with the development of Trypanosoma cruzi in these hosts.

Figure 3
Malformation of adult males of Rhodnius prolixus after continuous treatment with Z. caribaeum essential oil. (A) Normal legs of control insects (arrow), normal wings (asterisk). (B) and (C) Short wings (white arrowhead) and deformed (broad black arrow). Elongated legs (black arrowhead). Femur (a), tibia (b) and tarsus (c). Insect (B) shows an absence of the left glaw. (Nogueira et al. 2014a). The bar is equal to 1cm.

DYSDERCUS PERUVIANUS AND ONCOPELTUS FASCIATUS

Dysdercus peruvianus Guerin-Méneville (1831) (Heteroptera: Pyrrhocoridae) is popularly named cotton stainer bug. This insect is of great economic importance, being considered as the pest that causes serious economic damages in cotton plantations (Gossypium hirsutum L. - Malvaceae). Among damages, this phytophagous damage seeds and stain the cotton fibers with its waste, besides introducing fungi and bacteria (Chiang et al. 1970).

O. fasciatus (Dallas, 1852), belongs to the family Lygaoidea and, among the species of the genus Oncopeltus, it is undoubtedly one of the most used as a model in tests of both physiology and insect biochemistry, in addition to harboring protozoan species of the genus Phytomonas that cause damage to plant species of several families (Alves e Silva et al. 2013).

Both D. peruvianus and O. fasciatus (Fig. 4) are laboratory-friendly species that can be naturally found from the tropical regions of South America to the temperate ones in North America (Dingle et al. 1980, Dingle et al. 2003).

Figure 4
Adults of: (a) Dysdecus peruvianus and (b) Oncopeltus fasciatus. The bar is equal to 1.2 cm. Photos Saulo Rigon e Felipe Leite.

ORDER IXODIDA

The bovine tick Rhipicephalus (Boophilus) microplus (Canestrini, 1887) belongs to the phylum Arthropoda, class Arachnida, order Ixodida, subclass Acari, superfamily Ixodoidea and family Ixodidae (Figure 5). Cattle are the main host for this tick, though it also parasitizes sheep, horses, and even humans (Kerber et al. 2009). These species used are the only ones tested with extracts from the Restinga Plants by our group.

Figure 5
Tick Rhipicephalus (Boophilus) microplus: (a) eggs; (b) larvae; (c) adult females. Photos Camila Mattos.

This ectoparasite brings great damages to Brazilian agriculture; it is present in tropical and subtropical areas, being distributed throughout America, Africa, Asia and Australia (Mathison and Pritt 2014). In Brazil, this parasite is found at high levels throughout the year in the Southeast and Center-West regions (Figueiredo et al. 1999). Depending on the degree of infestation, the parasite can cause damage such as blood spoliation and anemia in the cattle and consequently weight loss, stress, decreased milk and meat production, as well as depreciation of the leather, since the parasite tears it to feed. In addition, perforations in the leather result in wounds that attract fly, compromising the quality of the material for the industry (Jonsson et al. 1998). Ectoparasitism is one of the major problems in the country’s milk and beef cattle production, causing damage to the animal’s health and consequent economic losses that reach $ 3.24 billion per year (Grisi et al. 2014, Figueiredo et al. 1999, Agnolin et al. 2008), especially the tick Rhipicephalus (Boophilus) microplus, responsible for 75% of these losses to bovine herds in Brazil. The tick, in addition to the irritating, hematophagic and toxic actions, can transmit protozoans responsible for the set of diseases called “Parasitic Bovine Sadness” (PBS). Economic losses are due to animal morbidity and mortality, reduced meat and milk production, abortions and reduced fertility (Grisi et al. 2014). Thus, an effective strategy to control the tick population could be to interfere with the cycles of egg laying while lowering the larval densities, and survival adults females. Several strategies are being developed in the control of cattle tick, although few are effective. (Kluck et al. 2018).

PLANTS USED AND MAIN RESULTS

The screening process based on the assessment of the biological activity of the different extracts, fractions, essential oils and secondary metabolites, indicated that five of the investigated plants presented IGRs endowed of the capacity to interfere in the life cycle of selected insects or mortality of adult females for ticks, these are: Eugenia sulcata Spring ex Mart. (Myrtaceae), Pilocarpus spicatus A.St.-Hil. (Rutaceae), Manilkara subsericea (Mart.) Dubard (Sapotaceae), Myrciaria floribunda (H.West ex Willd.) O.Berg (Myrtaceae) e Zanthoxylum caribaeum Lam. (Rutaceae) (Figure 6). A total of 100 secondary metabolites were identified in the essential oils for a total of 203 occurrences (Table I) and two triterpenes, a and b-amyrin acetates, from the EtOH crude extract of M. subsericea. The essential oil components are in their majority monoterpenes and sesquiterpenes, present either as hydrocarbons or oxygenated compounds. In E. sulcata, among the 37 identified components, sesquiterpenes largely dominate both in leaves essential oils (58.2%) as in stem oils (85.3%) (Lima et al. 2012). In Z. caribaeum, 54 compounds were identified: sesquiterpenes (47.3%) and monoterpenes (41.2%) are the major constituents (Nogueira et al. 2014a). In P. spicatus sesquiterpenes dominate (58.64%) the monoterpenes (36.93%) (Mello et al. 2007). On the contrary, in M. floribunda monoterpenes (53.9%) overcome sesquiterpenes (39.6%); monoterpenes are in their majority oxidized when sesquiterpenes are present mainly as hydrocarbons (Gonzalez et al. 2014).

Figure 6
Plants of Brazilian restinga vegetation. a-Eugenia sulcata, b-Myrciaria floribunda, c-Manilkara subsericea, d-Zanthoxylum caribaeum, e- Pilocarpus spicatus. Photos Marcelo Guerra Santos.

TABLE I
Composition of essential oils constituents of parts from: Eugenia sulcata, Myrciaria floribunda, Pilocarpus spicatus, Zanthoxylum caribaeum by GC/MS (% obtained by GC/FID peak-area normalization).sulcata

#	Compounds	Eugenia sulcata		Myrciaria floribunda			Pilocarpus spicatus	Zanthoxylum caribaeum
		Leaves	Stems	Leaves	Stems	Flowers	Leaves	Leaves
1	α-amorphene		0.7
2	δ-amorphene		4.0	4.2	1.4
3	aristolochene-4,5-di-epi							0.8
4	allo-aromadrendene		2.0
5	biciclogermacrene						6.2
6	α-bulsene							1.4
7	trans-cadina-1,4-diene	3.4	3.6					4.2
8	trans-cadina-1(6),4-diene					0.3
9	δ-cadinene						3.7
10	γ-cadinene	4.3	5.8				0.5
11	α-cadinol		2.3				0.9	1.7
12	epi-α-cadinol		1.1
13	trans-calamenene	4.4	7.0
14	camphene							0.4
15	camphoric acid		1.0
16	α-carene							3.7
17	trans-carveol							0.3
18	β-caryophyllene	24.7	18.8			3.3		4.3
19	(E)-caryophyllene				1.9		2.6
20	9-epi-E-caryophyllene		4.7			0.6
21	caryophyllene oxide	1.9	5.6
22	1,8-cineol	5.7
23	citronellal							0.3
24	citronellol							0.5
25	α-copaene	3,8	3.0		3.8		3.2
26	β-copaene	0.5	0.6			1.8
27	α-cubebene	1.2	1.1		1.7		0.4	0.4
28	cubebol							1.2
29	1-epi-cubebol					0.3
30	cubenol							0.6
31	1-epi-cubenol		1.7					0.9
32	b-cyclocitral							0.9
33	θ-cymene							0.5
34	dauca-5,8-diene		0.6				1.7
35	isodaucene							8.3
36	β-elemene		1.0			1.3		4.0
37	δ-elemene					0.2		0.7
38	elemol							0.8
39	eudesm-7(11)-en-4-ol				0.6
40	γ-eudesmol			3.3			0.3
41	10-epi-γ-eudesmol						0.3
42	geranyl acetate				1.5		0.9
43	(2E,6Z)-farnesol				1.4	0.8	16.1
44	(2E,6E)-farnesyl acetate					3.7	13.1	13.4
45	germacra-4(15),5,10(14)-trien-1α-ol				19.9			0.7
46	germacrene B					0.3		0.7
47	germacrene D	1.0	2.2
48	globulol		1.2
49	α-guaiene							1.4
50	guaiol					0.5
51	α-gurjunene					0.5
52	γ-gurjunene							0.6
53	n-heneicosane		0.4
54	hexanal							0.2
55	hexanol							0.4
56	n-hexanol							0.3
57	γ-himachalene	2.0	3.2		7.0
58	α-humulene	5.2	5.6	0.9	5.9		0.7	1,1
59	neo-intermedeol				0.8	1.1
60	ledol					1.7		0.8
61	limonene	1.1		2.6		1.6	14.7
62	linalool		0.5		2.6		12.7	2.0
63	p-trans-mentha-2-en-ol					7.0		0.3
64	cis-muurol-3,5-diene	0.6	0.9					0.6
65	trans-muurol-3,5-diene					2.7
66	cis-muurol-4(14),5-diene	1.3	1.1
67	trans-muurol-4 (14),5-diene					4.4		8.4
68	α-muurolene					0.8
69	γ-muurolene					7.0		0.9
70	α-muurolol					0.3		0.4
71	myltayl-4(12)-ene							0.4
72	myrcene	0.6		0.7		0.6	0.3	4.0
73	(E)-β-ocimene					0.1		2.0
74	(Z)-β-ocimene			0.8		7.6	0.3	0.4
75	(E)-ocimenone							0.4
76	β-phytol							1.0
77	α-pinene	17.3	0.3	0.4			5.1	7.6
78	β-pinene	11.0	0.3			1,4		0.7
79	pogostol				1.7
80	premnaspirodiene							0.8
81	sabinene					10.8		0.8
82	selin-11-en-4-α-ol		0.5
83	selina-3,7(11)-diene				3.8	4.6	1.6
84	α-7-epi-selinene					4.5		2.8
85	β-selinene	2.3	4.2		3.6
86	spathulenol	1.2	8.8			2.2		0.6
87	sylvestrene							11.3
88	α-terpinene			0.4		1.0		1.0
89	γ-terpinene			0.8		2.8	1.5	1.8
90	terpinen-4-ol			0.6		0.6	3.0	0.6
91	α-terpineol	1.3	0.6	5.5		5.4		1.0
92	terpinolene		2.7			2.0		0.4
93	n-tetradecanol		0.9
94	n-tricosane		0.3
95	α-tujene							0.3
96	valencene				1.8
97	viridiflorene							3.0
98	viridiflorol		1.4
99	zonarene			4.6		2.1	1.6	0.4
100	epi-zonarene	0.9	0.8			5.2
Number of identified metabolites		22	34	23	17	15	38	54

For several of the studied compounds, the biological activities observed are surprisingly high. One could observe increased mortality, molting and metamorphosis inhibition, paralysis, body deformities, the apparition of permanent nymphs, adultoids and juvenoids. Studies currently underway in our laboratories aim to define the chemical factors present in such extracts and their mechanism of action on the development and life cycle of these arthropods. The main results obtained are summarized in Table II.

TABLE II
Effects of extracts^I, essencial oils^II, sample fractions^III and purified metabolites^IV obtained from plants of Brazilian restinga vegetation on mortality, behavior and development of arthropods of medical or agricultural interest. The number under brackets after the plants species name represents the number of secondary metabolites detected in each plant.

Studied Plant [Reference]	Activities and Alterations
	Tested insect species
	Rhodnius prolixus	Oncopeltus fasciatus	Dysdercus peruvianus	Rhipicephalus microplus
Eugenia sulcata (37)II (Gonzalez et al. 2014)	NT	Mortality	Mortality	NT
Pilocarpus spicatus (17)II (Mello et al. 2007)	Mortality Behaviorb,c Developmentd,e	NT	NT	NT
Manilkara subsericea (2)I,III,IV (Fernandes et al. 2013)	NT	Mortality Developmentd,e,f,g	Mortality Developmentd,e,f,g	NT
Myrciaria floribunda (25)II (Tietbohl et al. 2014; Tietbohl et al - in press	Mortality Developmentf,g	Mortality	Mortality	NT
Zanthoxylum caribaeum (54)II(Nogueira et al. 2014a;Nogueira et al. 2014b)	Mortality Behaviorb Developmentg	NT	NT	Mortality of adult females Developmenth

• NT.: Not tested; Behavioral Changes: Lethargy^a, Paralysisb and Phagoinibition^c.; Developmental Activity: Delay in the period of molting^d, Molting inhibition (permanent nymphs)^e, Metamorphosis inhibition (overaged nymphs)^f, Morphological deformations^g and reduction of oviposition and hatching^h.

DISCUSSION

Currently, IGRs represent an important strategy for the control of important vectors in public health, veterinary medicine and agricultural pests due to their selective action, high environmental safety and low risk for non-target organisms, especially when compared to conventional insecticides/acaricides (Mazzonetto and Vedramim 2003, Isman 2006, WHO, 2006, Khan et al. 2016, Zaman et al. 2012).

Among plant components acting as IGRs, the essential oils play an outstanding role. They are complex mixtures containing many individual compounds, among them volatile terpenes and phenolic compounds, which are highly differentiated in different plant species (Regnault-Roger and Philogene, 2008). They are derived from aromatic plants that, throughout evolution, have developed such secondary metabolites that have helped attract pollinators or repel predatory insects (Walling, 2000).

Isolated constituents of essential oils also demonstrate efficacy against arthropods in laboratory or field conditions. Eugenol for example, abundant in Indian clove [Syzygium aromaticum (L.) Merr. & L.M.Perry – Myrtaceae], or cinnamaldehyde, abundant in cinnamon (Cinnamomum verum J.Presl - Lauraceae), exert toxic, ovicide, larvicide and adulticide activities on the Coleoptera Acanthoscelides obtectus (commonly known as the bean weevil) and inhibit its reproduction (Regnault-Roger et al. 1993). Eugenol is also highly toxic to mites Sarcoptes scabiei and Psoroptes cuniculi (Perrucci et al. 1995, Pasay et al. 2010), and is sold by a large number of suppliers under many different trade names for the domestic gardening market (Copping and Duke 2007).

In this sense, several products derived from plants also commercialized as insecticides and acaricides (George et al. 2008). Among the most remarkable is azadirachtin (extracted from seeds of Azadirachta indica A. Juss. - Meliaceae), nicotine (abundant in tobacco plants Nicotiana tabacum L. - Solanaceae), pyrethrin [extracted from the flower of Tanacetum cinerariifolium (Trevir.) Sch.Bip. – Asteraceae], rotenone (obtained from species of Derris, Lonchocarpus, and Tephrosia - all Fabaceae genera) among others (Schmeltz 1971). These used to control a wide range of arthropod pests including aphids, moths, beetles, fruit and vegetable mites, fire ants, mosquito larvae, lice, ticks and flies (Copping and Duke 2007). In developing countries, such products are traditionally used to control stored grain pests, such as coleopteran of the genus Sitophilus (De Oliveira et al. 2003), exhibiting low toxicity, easy biodegradation and species-specific selectivity (Borges et al. 2011, Vinturelle et al. 2017).

In our studies with metabolites obtained from 5 different plant genera of the Brazilian restinga vegetation, high biological activities were found involving increased mortality, inhibition of molting and metamorphosis, paralysis, body deformities, the appearance of overaged nymphs, extranumerary nymphs, adultoids and juvenoid, as well as partial or total inhibition of oviposition and egg hatching. These observations indicate the presence in obtained essential oils and purified fractions of chemical compounds capable of specifically interfering with the neuroendocrine system of arthropods by physiological routes related to the synthesis and release of vital hormones such as ecdysone (molt and metamorphosis inducing hormone) and juvenile hormone that controls, among others, of the reproductive and morphogenic processes. In some of the purified metabolites (i.e., α- and β-amyrin acetate) the presence of chitin synthesis inhibitory activity could also be inferred (Fernandes et al. 2013). Terpenes such as α- and β-amirin acetate is usually associated with interference with the neuroendocrine system and inhibition of moulting in arthropods (Regnault-Roger 1997). Other terpenes present in some of the essential oils used (i.e. α-pinene and β-pinene) were previously recognized for their insecticidal activity (Viegas-Junior 2003). Similarly, insecticidal activity related to inhibition of anticholinesterase activity in insects (Ryan and Byrne 1988) can be observed in some of the compounds analyzed here (Oliveira et al. 2010, Lima et al. 2012, Gonzalez et al. 2014). However, the isolated action of the purified molecules rarely reproduces the variety of biological effects reported for the use of extracts, fractions and essential oils. In addition, as a rule, the extracts and essential oils used show dose dependence biological effects, also indicative of a synergistic action between their different chemical constituents. From this perspective, the mode of action of the various secondary metabolites present in these compounds occurs as result of interactive mechanisms between them in the form of a vast network in which the metabolic pathways of insects and the molecules in the isolated fractions interact in a dynamic and flexible way. (Hummelbrunner and Isman 2001, Rattan 2010, Rizzati et al. 2016). In this way, the presence of various substances in these complex mixtures is likely to increase their effectiveness as insecticides/acaricides and reduce the evolution of resistance to these natural insecticides when compared to the purified molecules present in commercial insecticides, as reported for Myzus persicae – Hemiptera (Feng and Isman 1995, Isman 2006). These compounds are now being studied further in our laboratories to determine if they may, or may not, be useful in controlling insect populations and/or interfere with the life cycle and vector transmission of parasites.

CONCLUSIONS

For several of the compounds studied, the biological activities (insecticide and acaricide) found were surprisingly high and are undergoing further chemical and physiological studies to determine if these substances may or not be useful in controlling arthropods and vector transmission in the field. In addition, several of the collected botanical specimens are still the subject of further screening studies, which provide us with a perspective of long-term productive continuous supply of new information on the systems studied.

Crude extracts, secondary metabolites and essential oils obtained from plants in the Brazilian restinga vegetation, exhibit high activity as development regulators in arthropods and present viability for their use in integrated programs of ecological control of populations of insects and ticks of interest in medicine and agriculture.

REFERENCES

• AGNOLIN FL, LUCERO SO and BOGAN S. 2008. Galea musteloides en la provincia de Santa Cruz, Argentina. Mastozool Neotrop 15(1): 113-115.
• ALVES E SILVA TL, VASCONCELLOS LRC, LOPES AH and SOUTO-PADRÓN. 2013. The Immune Response of Hemocytes of the Insect Oncopeltus fasciatus against the Flagellate Phytomonas serpens. Plos One 8(8): 72076.
• ARAUJO DSD. 1992. Vegetation types of sandy coastal plains of tropical Brazil: a first approximation. In: Coastal Plant Communities Of Latin Americ. San Diego: U. SEELIGER, p. 337-347.
• ARAÚJO DSD. 2000. Análise Florística e Fitogeográfica das Restingas do Estado do Rio de Janeiro. Tese de Doutorado, Universidade Federal do Rio de Janeiro, Rio de Janeiro.
• ARAUJO DSD and HENRIQUES RPB. 1984. Análise florística das restingas do Estado do Rio de Janeiro. In: Lacerda LD, Araujo DSD, Cerqueira R and Turcq B (Eds), Restingas: origem, estrutura, processos, Niteroi: CEUFF, p. 159-193.
• AZAMBUJA P, GARCIA ES, WANIEK PJ, VIEIRA CS, FIGUEIREDO MB, GONZALEZ MS, MELLO CB, CASTRO DP and RATCLIFFE NA. 2017. Rhodnius prolixus: from physiology by Wigglesworth to recent studies of imune system modulation by Trypanosoma cruzi and Trypanosoma rangeli. J Ins Physiol 97: 45-65.
• BARROS GSC, ADAMI ACO and ZANDONÁ NF. 2014. Faturamento e volume exportado do agronegócio brasileiro são recordes em 2013. CEPEA – ESALQ/USP.
• BEATY BJ and MARQUARDT WC 1996. Fleas and the agents they transmit. In: Thomas RE (Ed), The Biology Of Disease Of Vectors. University Press of Colorado, p. 146-159.
• BORGES LM, FERRI PH, SILVA WJ, SILVA WC and SILVA JG. 2003. In vitro efficacy of extracts of Melia azedarach against the tick Boophilus microplus. Med Vet Entomol 17: 228-231.
• BORGES LMF, SOUSA LAD DE and BARBOSA CDS. 2011. Perspectives for the use of plant extracts to control the cattle tick Rhipicephalus (Boophilus) microplus. Braz J Vet Parasitol 20: 89-96.
• CAMPANHOLA C. 2005. Avanços na pesquisa agropecuária brasileira. Revista USP 64: 68-75.
• CHAGAS C. 1909. Nova tripanozomiase humana: estudos sobre a morfolojia e o ciclo evolutivo do Schizotrypanum cruzi n. gen., n. sp., ajente etiolojico de nova entidade morbida do homem. Mem Inst Oswaldo Cruz Rio de Janeiro 1(2):159-218.
• CHAGAS C. 1911. Moléstia de Carlos Chagas. II Conferência realizada na Academia Nacional de Medicina (1911). In: Prata AR (Ed), Carlos Chagas, Coletânea De Trabalhos Cientificos Brasilia: Editora Universidade de Brasília, p. 167-192.
• CHIANG HC, STERN VM and CHIARA PPA L. 1970. The FAO Manual on Crop Losses Due to Agricultural Pests, With an Invitation to U. S. Entomologists to Contribute Information. Bull Entomol Soc America 16(2): 83-84.
• COPPING LG and DUKE SO. 2007. Natural products that have been used commercially as crop protection agents. Pest Manag Sci 63(6): 524-54.
• CORTEZ MGR, GONZALEZ MS, CABRAL MMO, GARCIA ES and AZAMBUJA P. 2002. Dynamic development of Trypanosoma cruzi in Rhodnius prolixus: role of decapitation and ecdysone therapy. Parasitol Res 88: 697-703.
• DE OLIVEIRA S, VENDRAMIM PJ, RIBEIRO JÚNIOR JI and SANTOS JB. 2003. Bioactivity of powders from some plants on Sitophilus zeamais Mots. (Coleoptera: Curculionidae). Ciênc Agrotec 1231-1236.
• DINGLE H, ALDEN BM, BLAKLEY NR, KOPEC D and MILLER ER. 1980. Variation in photoperiodic response within and among species of milkweed bugs (Oncopeltus). Evolut 34 (2): 356-370.
• DINGLE H, BLAKLEY NR and MILLER ER. 2003. Variation in body size and flight performance in milkweed bugs (Oncopeltus). Evolut 34(2): 371 -385.
• FAO STATICAL YEAR BOOK. 2013. World Food and Agriculture. Food and Agriculture Organization of the United Nations. Rome.
• FARIA ABC. 2009. Revisão sobre alguns grupos de inseticidas utilizados no manejo integrado de pragas florestais. Ambiência. Rev do Setor de Ciências Agrárias e Amb 5(2): 358.
• FARIA NMX, FASSA ACG and FACCHINI LA. 2007. Pesticides poisoning in Brazil: the official notification system and challenges to conducting epidemiological studies. Ciênc Saúde Colet 12 (1): 25-38.
• FEDER D, VALLE D, REMBOLD H and GARCIA ES. 1988. Azadiracthtin induced sterilization in mature female of Rhodnius prolixus. Zeitschrift fur Natur 43: 908-913.
• FENG R. and ISMAN MB. 1995. Selection for resistance to azadirachtin in the green peach aphid, Myzus persicae. Exper 51(8): 831-833.
• FERNANDES CP, XAVIER A, PACHECO JPF, SANTOS MG, MEXAS R, RATCLIFFE NA, GONZALEZ MS, MELLO CB, ROCHA L, FEDER D. 2013. Laboratory evaluation of the Manilkara subsericea (Mart.) Dubard extracts and triterpenes on development of Dysdercus peruvianus and Oncopeltus fasciatus. Pest Manag Sci 69: 292-301.
• FIGUEIREDO LTM, BADRA SJ, PEREIRA LE and SZABÓ MPJ. 1999. Report on ticks collected in the Southeast and Mid-West regions of Brazil: analyzing the potential transmission of tick-borne pathogens to man. Rev. Soc Bras Med Trop 32 (6): 613-619.
• GARCIA ES and AZAMBUJA P. 1991. Development and interactions of Trypanosoma cruzi within the insect vector. Parasitol Tod 7: 240-244.
• GARCIA ES and AZAMBUJA P. 2004. Lignoids in insects: chemical probes for study of moulting, excretion and Trypanosoma cruzi–triatomine interactions. Toxicon 44: 431-440.
• GEORGE DR, GUY JH, ARKLE S, HARRINGTON D, LUNA C, OKELLO EJ, SHIEL RS, PORT G, SPARAGANO OAE. 2008. Use of Plant-derived Products to Control Arthropods of Veterinary Importance: A Review. In: Ann NY (Ed), Animal Biodiversity And Emerging Diseases. Acad Sci 1149, p. 23-26.
• GONZALEZ MS, LIMA BG, OLIVEIRA AFR, NUNES D, FERNANDES CP, GUERRA MS, TIETBOHL LAC. MELLO CB, ROCHA L and FEDER D. 2014. Effects of essential oil from leaves of Eugenia sulcata on the development of agricultural pest insects. Rev Bras Farmacogn 24: 413-418.
• GONZALEZ MS, NOGUEIRA NFS, DE SOUZA W, AZAMBUJA P and GARCIA ES. 2000. Influence of brain on the Trypanosoma cruzi development in Rhodnius prolixus. Mem Inst Oswaldo Cruz 95: 58.
• GRAF JF. 1993. The role of insect growth regulators in arthropod control. Parasitol Tod 9: 471-474.
• GRISI L, LEITE RC, MARTINS JRS, BARROS ATM, ANDREOTTI R, CANÇADO PHD, LEON AP, PEREIRA JB and VILLELA HS. 2014. Reassessment of the potential economic impact of cattle parasites in Brazil. Bras J Veter Parasitol 23: 150-156.
• GULLAN PJ and CRANSTON PS. 2008. The Insects: An Outline of Entomology. Entomologia. 3. ed. Department of Entomology, University of California, Davis, EUA: Roca, 427.
• HEMINGWAY J and RANSON, H. 2000. Insecticide Resistance in Insect Vectors of Human Disease. Annu Rev Entomol 45: 371-391.
• HUMMELBRUNNER LA and ISMAN MB. 2001. Acute, sublethal, antifeedant, and synergistic effects of monoterpenoid essential oil compounds on the tobacco cutworm, Spodoptera litura (Lep., Noctuidae). J Agric Food Chem 49:715-20.
• ICMBIO. 2017. Instituto Chico Mendes de Conservação da Biodiversidade 2016. Parque Nacional de Jurubatiba. Disponível em: http://www.icmbio.gov.br/ parnajurubatiba/. Last access: 29/11/2017.
  » http://www.icmbio.gov.br/
• ISMAN MB. 2006. Botanical insecticides, deterrents and repellents in modern agriculture and an increasingly regulated world. Annu Rev Entomol 51: 45-66.
• JAYARAJ R, MEGHA P and SREEDEV P. 2016. Organochlorine pesticides, their toxic effects on living organisms and their fate in the environment. Interdiscip Toxicol 9(3-4): 90-100.
• JONSSON NN, MAYER DG, MATSCHOSS AL, GREEN PE and ANSELL J. 1998. Production effects of cattle tick (Boophilus microplus) infestation on high yielding dairy cows, draft paper for Veterinary Parasitol 78 (1): 65-77.
• KERBER CE, LABRUNA MB, FERREIRA F, DE WAAL DT, KNOWLES DP and GENNARI SM. 2009. Prevalence of equine Piroplasmosis and its association with tick infestation in the State of São Paulo, Brazil. Vet Jaboticabal 18: 1-8.
• KELECOM A, REIS GL, FEVEREIRO PCA, SILVA JG, SANTOS MG, MELLO, CB, GONZALEZ MS, GOUVEA RSC, ALMEIDA GSS. 2002a. A multidisciplinary approach to the study of the Fluminense vegetation. An Acad Bras Cienc 74: 171-181.
• KELECOM A, ROCHA MA, MAJDALANI EC, GONZALEZ MS, MELLO CB. 2002b. Novas atividades biológicas em antigos metabólitos: ácido oleanóico e eugenol de Eugenia caryophyllata. Braz J Pharmacol 12: 70-71.
• KHAN GZ, KHAN I, KHAN IA, ALAMZEB, SALMAN M, ULLAH K. 2016. Evaluation of different formulations of IGRs against Aedes albopictus and Culex quinquefasciatus (Diptera: Culicidae). Asian Pacific J Tropical Biom 6(6): 485-491.
• KLUCK GEG, CARDOSO LS, DE CICCO NNT, MICHELE S, LIMA MS, FOLLY E and ATELLA GC. 2018. A new lipid carrier protein in the cattle tick Rhipicephalus microplus. Ticks and Tick-borne Dis 9: 850-859.
• KOGAN M. 1998. Integrated Pest Management: Historical Perspectives and Contemporany Development. Ann Rev Entomol 43: 243-270.
• KOLLIEN AH and SCHAUB GA. 2000. The development of Trypanosoma cruzi in Triatominae. Parasitol Today 16: 381-387.
• LIMA BG, TIETBOHL LAC, FERNANDES CP, CRUZ RAS, BOTAS GS, SANTOS MG, SILVA-FILHO MV and ROCHA LM. 2012. Chemical composition of essential oils and anticholinesterasic activity of Eugenia sulcata Spring ex Mart. Lat Am J Pharm 31: 152-155.
• MAGNAGO LFS, MARTINS SV and OBERDAN JP. 2011. Floristic heterogeneity of phytocenoses restinga in Rio de Janeiro and Espírito Santo states, Brazil. Rev Árvore 35 (2): 245-254.
• MARQUES MCM and OLIVEIRA PEAM. 2004. Fenologia de espécies do dossel e do sub-bosque de duas Florestas de Restinga na Ilha do Mel, sul do Brasil. Rev Bras Bot 27 (4): 713-723.
• MATHISON BA and PRITT BS. 2014. Laboratory Identification of Arthropod Ectoparas Clin Microbiol Rev 27: 648-667.
• MAZZONETTO F and VEDRAMIM JD. 2003. Effect of Powders from Vegetal Species on Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae) in Stored Bean. Neotrop Entomol 32(1): 145-149.
• MELLO CB, MENDONÇA-LOPES, FEDER D, UZEDA CD, CARNEIRO RM, ROCHA MA and GONZALEZ MS. 2008. Laboratory evaluation of the effects of triflumuron on the development of Rhodnius prolixus nymph. Mem Inst Osw Cruz 103: 839-842.
• MELLO CB, UZEDA CD, BERNARDINO MV, MENDONÇA–LOPES D, KELECOM A, FEVEREIRO PCA, GUERRA MS, OLIVEIRA AP, ROCHA LM and GONZALEZ MS. 2007. Effects of the essential oil obtained from Pilocarpus spicatus Saint – Hilaire (Rutaceae) on the development of Rhodnius prolixus ninphae. Braz J Pharmacol 14: 514 - 521.
• MONDAL K and PARWEEN S. 2001. Insect Growth regulators and their potential in the management of stored-product insect pests. Integrat Pest Managem 5: 255-295.
• NOGUEIRA J, MOURÃO SC, DOLABELA IB, SANTOS MG, MELLO CB, KELECOM A, MEXAS R, FEDER D, FERNANDES CP, GONZALEZ MS and ROCHA L. 2014a. Zanthoxylum caribaeum (Rutaceae) essential oil: chemical investigation and biological effects on Rhodnius prolixus nymph. Parasitol Res 113: 4271-4279.
• NOGUEIRA J, VINTURELLE R, MATTOS C, TIETBOHL LAC, SANTOS MG, JUNIOR ISV, MOURÃO SC, ROCHA L, FOLLY E. 2014b. Acaricidal Properties of the Essential Oil from Zanthoxylum caribaeum against Rhipicephalus microplus. J Med Entomol 51(5): 971-975.
• OLIVEIRA AP, CRUZ RAS, BOTAS GS, GONZALEZ MS, SANTOS MG, TEIXEIRA LA, ROCHA LM. 2010. Chemical and Biological Investigations of Pilocarpus spicatus essential oils. Boletín Latin American y del Caribe de Plantas Medicinales y Aromáticas 9(3): 206-211.
• OPAS. 2014. Homepage: http://www.paho.org/hq/index.php?option=com_topics&view=article&id=10&Itemid=40743&lang=es Acess in 21/10/2017.
  » http://www.paho.org/hq/index.php?option=com_topics&view=article&id=10&Itemid=40743&lang=es
• PASAY C, MOUNSEY K, STEVENSON G, DAVIS R, ARLIAN L, MORGAN M, VYSZENSKI-MOHER D, ANDREWS K and MCCARTHY J. 2010. Acaricidal Activity of Eugenol Based Compounds against Scabies Mites. PLoS ONE 5(8): 12079.
• PERRUCCI S, MACCHIONI G, CIONI PL, FLAMINI G and MORELLI I. 1995. Structure/activity relationship of some natural monoterpenes as acaricides against Psoroptes cuniculi. J Nat Prod 58: 1261-1264.
• RAJPUT ZI, HU SH, CHEN WJ, ARIJO AG and XIAO CW. 2006. Importance of ticks and their chemical and immunological control in livestock. J Zhejiang Univ Sci B 7(11): 912-921.
• RATTAN RS. 2010. Mechanism of action of insecticidal secondary metabolites of plant origin. Crop Protect 29: 913-920.
• REGNAULT-ROGER C. 1997. The potential of botanical essential oils for insect pest control. Integ Pest Manag Rev 2: 25-34.
• REGNAULT-ROGER C, HAMRAOUI A, HOLEMAN H, THERON E and PINEL R. 1993. Insecticidal effect of essential oils from mediterranean plants upon Acanthoscelides Obtectus Say (Coleoptera, Bruchidae), a pest of kidney bean (Phaseolus vulgaris L.). J of Chem Ecol 19(6): 1233-1244.
• REGNAULT-ROGER C and PHILOGÈNE BJR. 2008. Past and current prospects for the use of botanicals and plant allelochemicals in integrated pest management. Pharm Biol 46: 41-51.
• RIZZATI V, BRIAND O, GUILLOU H and GAMET-PAYRASTRE L. 2016. Effects of pesticide mixtures in human and animal models: An update of the recent literature. Chem Biol Interact 254: 231-246.
• ROCHA L, SILVA V, MOACÉLIO V, SANTOS MG, BOTAS GS, CRUZ RAS, FERNANDES CP, TIETBOHL LAC and LIMA BG. 2012. Chemical composition of essential oils and anticholinesterasic activity of Eugenia sulcata spring ex mart. Latin Am J Pharmacy 31: 152-155.
• ROSADO BHP and DE MATTOS EA. 2007. Variação temporal de características morfológicas de folhas em dez espécies do Parque Nacional da Restinga de Jurubatiba, Macaé, RJ, Brasil. Acta Botan Brasil 21(3):741-752.
• RYAN MF and BYRNE O. 1988. Plant-insect coevolution and inhibition of acetylcholinesterase. J Chem Ecol 14: 1965-1975.
• SÁ CFC. 2014. Riqueza, conservação e conhecimento tradicional de plantas das restingas fluminenses. In: Oliveira AP, Rocha LM, Santos MG (Eds), III Simpósio Flora Das Restingas Fluminenses: Potencial Econômico Econservação. http://docs.wixstatic.com/ugd/fec81a_efdf46eb58ae4861b21f2c709993af63.pdf. Access 01/12/2017.
  » http://docs.wixstatic.com/ugd/fec81a_efdf46eb58ae4861b21f2c709993af63.pdf.
• SANTOS MG, FEVEREIRO PCA, REIS GL and BARCELOS JI. 2009. Recursos vegetais da Restinga de Carapebus, Rio de Janeiro, Brasil. Rev Biol Neotrop 6: 35-54.
• SANTOS MG, SYLVESTRE LS and DE ARAUJO DSD. 2004. Análise florística das pteridófitas do Parque Nacional da Restinga de Jurubatiba, Rio de Janeiro, Brasil. Acta Bot Bras 18(2): 271-280.
• SCHMELTZ I. 1971. Naturally Occurring Insecticides. New York. M. Jacobson and D.G. Crosby, D.G. Marcel Dekker.
• SMITH CD and RAUPP MJ. 1986. Economic and Environmental Assessment of an Integrated Pest Management Program for Community-owned Landscape Plants. J Econ Entomol 79: 162-165.
• TAIZ L and ZEIGER E. 1991. Plant Physiology. Benjamin/Cummings Pub. Co., Science, 559 p.
• TIETIBOHL LAC et al. 2014. Laboratory evaluation of the effects of essential oil of Myrciaria floribunda leaves on the development of Dysdercus peruvianus and Oncopeltus fasciatus. Rev Bras Farmac 24: 316-321.
• TIETBOHL LAC, BARBOSA T, FERNANDES CP, SANTOS MG, SILVA FEB, DENARDIN ELG, BACHINSKI R, ALVES GG, SILVA-FILHO V and ROCHA L. 2012 (b) Comparative study and Anticholinesterasic evaluation pf essential oils from leaves, stems and flowers of Myrciaria floribunda (H. West ex Willd.) O. Berg. Latin Americ J Pharmacy 31(4): 637-641.
• VIEGAS JUNIOR C. 2003. Terpenos com atividade inseticida: uma alternativa para o controle químico de insetos. Quim. Nova 26: 390-400.
• VINTURELLE R, MATTOS C, MELONI J, NOGUEIRA J, NUNES MJ, VAZ JR ISV, ROCHA L, LIONE, V, CASTRO HC and CHAGAS EF. 2017. In Vitro Evaluation of Essential Oils Derived from Piper nigrum (Piperaceae) and Citrus limonum (Rutaceae) against the Tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Biochem Res Inter 1-9.
• WALLING LL. 2000. The myriad plant responses to herbivores. J Plant Growth Regul 19: 195-216.
• WHO - WORLD HEALTH ORGANIZATION. 2006. Pesticides and their application: for the control of vectors and pests of public health importance. WHO, Genebra.
• ZAIM M and GUILLET P 2002. Alternative insecticides: an urgent need. Trends in Parasitol 18 (4): 161-163.
• ZAMAN MA, IQBAL Z, ABBAS RZ, KHAN MN, MUHAMMAD G, YOUNUS M, AHMED S. 2012. In vitro and in vivo acaricidal activity of a herbal extract.Vet. Parasitol 186: 431- 436.

Publication Dates

• Publication in this collection
  08 Apr 2019
• Date of issue
  2019

History

• Received
  20 Jan 2018
• Accepted
  25 July 2018