Larvicidal Activity of Eucalyptus globulus ( Myrtaceae ) and Origanum majorana ( Lamiaceae ) Essential Oils against the Sand Fly Phlebotomus papatasi ( Diptera : Psychodidae )

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INTRODUCTION
Phlebotomine sand flies are the vectors of medically important viruses, the bacterium Bartonella bacilliformis, and most importantly, the protozoan parasites that cause leishmaniasis (WHO, 2008).Sand flies also are often significant biting flies of man; it has been documented that their bites can cause acute dermatitis and delayed-type hypersensitivity reactions (Belkaid et al., 2000).
Phlebotomus papatasi (Diptera: Psychodidae) is the vector of Leishmania major, the causative agent of zoonotic cutaneous leishmaniasis (ZCL) throughout North Africa, the Middle East, Southwest Asia (WHO, 2008), andin North Sinai, Egypt (Fryauff et al., 1993).One of the methods to interrupt disease transmission is to control the vectors.Chemical insecticides play a major role in efforts to manage vector populations.Although chemicals have been used successfully as components of pest management strategies, many of these synthetic insecticides promoted wide spread contamination of the environment, toxicity to non-target organisms, resistance development and destructive effects on human and animal health.An alternative and recent approach for control is the use of natural products of plant origin known as botanical derivatives (George et al., 2014).
Plant essential oils (EOs) are lipophilic secondary metabolites of which terpenoids are the main component.Many studies showed neurotoxic action of EOs in insects.The inhibition of acetylcholine esterase (AChE) is the most investigated mechanism of EOs action.Other proposed mechanism of EOs action is a positive allosteric modulation of gamma-aminobuteric acid (GABA) receptors.Essential oils act also via the octopaminergic system.Disruption of octopamine results in the total breakdown of the nervous system in insects.The lack of octopamine receptors in vertebrates provides the mammalian selectivity of EOs as insecticides.A multitude of potential targets in the insect nervous system makes EO components interesting candidates for bioinsecticides (Jankowska et al., 2018).
Eucalyptus is one of the most cultivated genera in the world, including more than 700 species belonging to Myrtaceae family.
Various biological properties have already been attributed to the genus Eucalyptus, among them the repellent action against Phlebotomus papatasi (Yaghoobi-Ershadi et al., 2006), and insecticidal activity on the different developmental stage of Lutzomyia longipalpis in the laboratory (Maciel et al., 2010).Eucalyptus oil has been placed under generally regarded as safe (GRAS) category by Food and Drug Authority of USA and classified as non-toxic (USEPA, 1993).
The genus Origanum is composed of 42 species widely distributed in North Africa (Ietswaart, 1980).These species are used since the ancient times as spices, medicinal, aromatic and ornamental plants (Meyers, 2005).Literature data related to Origanum majorana bioactivities are limited.El Idrissi et al. (2014) assessed the fumigant action of the EO of O. majorana against the weevil Bruchus lentis on lentil seeds and recommended the use of oils in integrated management of weevils.Mossa and Nawwar (2010) highlighted that O. majorana EO was found to be promising natural sources of bioactive compounds due to its high phenolic and flavonoid contents.Duletić-Laušević et al. (2018) found that among tested marjoram samples originated from Serbia, Greece, Egypt and Libya, the most promising were plants from Serbia and Egypt which contained high level of rosmarinic acid.This acid was found to predominant constituent in the extract and presumably had substantial influence on their antioxidant and antineourogenerative activities.
Eucalyptus globulus (also called the blue gum) and Origanum majorana (also called marjoram) are planted in Egypt.Accordingly, this study investigated for the first time the larvicidal activity of EOs of these two plants on the sand fly Phlebotomus papatasi.

MATERIAL AND METHODS Plant Oils:
Two essential oils were used in this study; Eucalyptus globulus and Origanum majorana, (Sigma-Aldrich, St. Louis, MO, USA).

Sand Flies:
The sand fly species used in this investigation was a laboratory bred Phlebotomus papatasi.Colonization and rearing procedures were those of Modi and Tesh (1983).Sand flies were reared in a walk-in insectary environmentally controlled and regulated to 272°C and 60-70 % relative humidity, and 12:12 (L:D) photoperiod.Adults were fed on 30 % sucrose solution and on hamster as a source of blood for females.Larval food consisted of dried rabbit pellets and cow blood; grinded and autoclaved.

Larvicidal Bioassays:
The larvicidal activity of Eucalyptus globulus and O. majorana EOs were carried out against third instar larvae of Phlebotomus papatasi.Ten larvae were added to a 7-dram polystyrene vial with a bottom layer of 2 cm plaster of Paris containing 0.5 mg larval food.
The control group included ten larvae sprayed with 1 ml Tween 80 (3 %).Three replicates for each treatment was carried out.Larval mortalities were recorded after 48 h and data obtained from each oil concentration were subjected to regression analysis by probit to generate values for LC 50 and LC 90 using the software package of IBM SPSS Statistics version 20 (SPSS Inc., Chicago, Il, USA).

Scanning Electron Microscopy:
Normal and larvae treated with LC 50 were washed in saline solution, then fixed in 70% alcohol.They were mounted on aluminum stubs with double-sided clear adhesive tape and sputter-coated on an Eduardo S150 Sputter coater with a thinlayer of gold.Specimens were examined for image analysis with JEOL-JSM-5500LV with high vacuum mode at the Regional Center of Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt.

Histology:
To investigate the changes in midgut cells, larvae were exposed to LC 50 of EOs of E. globulus and O. majorana for 48 h.Paraffin sections 6 μm thin, were stained with hematoxylin and eosin (HE) and examined microscopically (X400).

RESULTS AND DISCUSSION Larval Bioassay:
Larvicidal activity of E. globulus and O. majorana essential oils was found to be dependent upon oil concentrations tested.Lethal concentration, LC 50 of E. globulus oil was 677 g/ml; while LC 90 was 928 g/ml.The LC 50 value for O. majorana was 91g/ ml; while LC 90 was 142 g/ml (Table 1).From such results, O. majorana essential oil was more active against the sand fly larvae based on its lower LC 50 value.The treated larvae tended to show toxic symptoms including hyperactivity, convulsions and tremors followed by paralysis and death.
Data on the larvicidal bioactivity of EOs especially on the sand fly larvae are scarce.We attempted to compare the lethal dose obtained in the present study with those reported in the literature.Only one study on the New world sand fly Lutzomyia longipalpis by Maciel et al. (2010) was available.The authors found that Eucalyptus EOs were effective on the three phases of the insect (egg, larva and adult) and E. staigeriana was the most effective followed by E. citriodora and E. globulus.Eucalyptus golobulus showed 100% effectiveness at 40 mg/ ml, while E. citriodora and E. staigeriana oils showed this result at lower concentrations of 6.5 and 5 mg/ml respectively.Our results for E. globulus on P. papatasi larvae were much less than that obtained for Lu.longipalpis larvae.
Available literature on the activity of O. majorana oil and its components showed fumigant action, ovicidal and adulticidal activity against pests of stored products (e.g.El Idrissi et al., 2014).
Recently, mosquito control programs target the elimination of mosquito larval stages at their breeding sites with larvicides since adulticides may reduce the adult population only temporarily (Chung et al., 2009;Conti et al., 2010).To apply this approach to control sand flies, more conclusive studies to locate their breeding sites are required.

Scanning Electron Microscopy (SEM):
To visualize the effect of oil treatment on the larval surface, scanning electron microscopy was employed.Figures 1, 2 and 3 compare the structural changes in larvae treated with LC 50 of each EOs and untreated ones.E. globulus oil-treated larvae (Fig. 2) showed significant shrinkage of intersegmental region, distortion of microtrichia (m) (Fig. 2A and B), malformation or degeneration of anterior spiracle (Fig. 2C) as compared to the untreated ones (Fig. 1); in addition to partial closure of posterior spiracle (Fig. 2D).
wrinkled cuticular surface of the head and thorax (Fig. 3B).The abdominal segments showed distortion of microtrichia and squamiform sensilla (Fig. 3C). Figure 3D, highlights deformed and complete closure of the abdominal spiracles.
The above observations indicated that E. globulus and O. majorana oils caused substantial damages on the surface of sand fly larvae.
A clear damage was also observed on the spiracles which suffered degeneration, deformation and partial or complete closure.It is hypothesized that the EO causes intoxification to the larvae, and in response the larvae close the spiracles on the abdominal segments as a detoxification process.

Histological Analysis:
Histology data of larvae treated with LC 50 of E. globulus was performed 48 h post exposure to confirm histological changes.In the control group, the midgut tissue is formed of columnar epithelial cells arranged in a single layer and a well-developed brush border.The epithelium along the midgut rested on non-cellular basal lamina, under which there is a thin musculature consisting of inner circular and outer longitudinal muscles.The peritrophic matrix is present as a thin thread like membrane within the midgut lumen (Fig. 4A).In the larvae exposed to E. globulus (Fig. 4B), the gut cells were detached from the basal lamina and were separated from each other leaving clear gaps.Marked blebbing and release of cell content in the lumen was also observed.The brush border increased in size reaching almost the length of the epithelial cells.Disintegration of the peritrophic matrix and release of the food particles in the ectoperitrophic space was noticed.
When the third instar larvae of P. papatasi were exposed to O. majorana (Fig. 4C), the columnar cells exhibited marked elongation and development of protrusions growing out towards the gut lumen.Outgrowth of the brush border, degeneration of the peritrophic matrix was observed.
The mechanism of action of EOs on larvae has not been clearly elucidated.Although its larvicidal activity was reportedly mediated by their comparative C absorption to larvae midgut (Rey et al., 1999) and neurotoxicity potential (Coats et al., 1991).Midgut cells have been described as a possible target for plant-derived compounds, resulting in various midgut disorders and destruction of brush border (Ribeiro-Neto et al., 2017).Treatment of P. papatasi larvae with both EOs resulted in the degeneration of the peritrophic matrices thus releasing midgut content into the lumen and the ectoperitrophic space.The peritrophic matrices are considerable physical barriers between the contents of the insect-gut lumen and the underlying digestive epithelial cells of the insect midgut (Tellam, 1996).The observed histopathological effect on the midgut of P. papatasi larvae due to E. globulus and O. majorana oils were similar to the effect of other xenobiotics which triggered a detoxification process (Alves et al., 2018).
Regardless of the type of substance used, these changes were common response to cellular intoxication.
Results obtained by E. globulus and O. majorana EOs shed light on their possible role in developing eco-friendly product for sand fly control.The EOs mechanism of action is not well established, however, toxicity by contact might be reasonable way of action.

Table 1 :
Mean percent mortality of Phlebotomus papatasi third instar larvae treated with Eucalyptus globulus and Origanum majorana essential oils