Mosquito Larvicidal Activity, Antimicrobial Activity, and Chemical Compositions of Essential Oils from Four Species of Myrtaceae from Central Vietnam.

Mosquitoes are important vectors of several diseases, and control of these insects is imperative for human health. Insecticides have proven useful in controlling mosquito populations, but insecticide resistance and environmental concerns are increasing. Additionally, emerging and re-emerging microbial infections are problematic. Essential oils have been shown to be promising mosquito larvicidal agents as well as antimicrobial agents. In this work, the essential oils from four species of Myrtaceae (Baeckea frutescens, Callistemon citrinus, Melaleuca leucadendra, and Syzygium nervosum) growing wild in central Vietnam have been obtained by hydrodistillation and analyzed by gas chromatographic techniques. The essential oils have been screened for mosquito larvicidal activity against Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus, and for antimicrobial activity against Enterococcus faecalis, Staphylococcus aureus, and Candida albicans. Callistemon citrinus fruit essential oil, rich in α-pinene (35.1%), 1,8-cineole (32.4%), limonene (8.2%), and α-terpineol (5.8%) showed good larvicidal activity with 24-h LC50 = 17.3 μg/mL against both Ae. aegypti and Cx. quinquefasciatus, and good antibacterial activity against E. faecalis (minimum inhibitory concentration (MIC) = 16 μg/mL) The 48-h larvicidal activities of M. leucadendra leaf essential oil, rich in α-eudesmol (17.6%), guaiol (10.9%), linalool (5.1%), (E)-caryophyllene (7.0%), and bulnesol (3.6%) were particularly notable, with LC50 of 1.4 and 1.8 μg/mL on Ae. aegypti and Cx. quinquefasciatus. Similarly, M. leucadendra bark essential oil, with α-eudesmol (24.1%) and guaiol (11.3%), showed good antibacterial activity against. E. faecalis. Both B. frutescens and C. citrinus leaf essential oils demonstrated anti-Candida activities with MIC values of 16 μg/mL. The results of this investigation suggest that essential oils derived from the Myrtaceae may serve as “green” alternatives for the control of mosquitoes and/or complementary antimicrobial agents.


Introduction
Mosquitoes are important vectors of diseases and kill more humans than any other animal. Aedes aegypti (L.) and Ae. albopictus (Skuse) (Diptera: Culicidae) are vectors of the yellow fever, dengue, Zika, and chikungunya viruses [1][2][3]; Culex quinquefasciatus (Say) is the primary vector of the Saint Louis encephalitis and West Nile viruses, as well as the filarial nematode Wuchereria bancrofti, and may also be a vector of the Zika virus [4].
Microbial infections continue to be a problem, for humans [5], as well as for livestock and other agriculture settings [6][7][8]. Compounding this problem are newly emerging pathogenic microorganisms, in addition to re-emerging multidrug-resistant pathogens [9,10].
The Myrtaceae is comprised of 131 genera and around 5500 species, all of which are woody trees or shrubs and contain essential oils [11]. Several members of the family are commercially important for their medicinal essential oils, such as clove (Syzygium aromaticum (L.) Merr. & L.M. Perry), tea tree (Melaleuca alternifolia Cheel), allspice (Pimenta dioica (L.) Merr.), and Eucalyptus. In this work, we present the essential oil compositions of four species of Myrtaceae growing wild in central Vietnam, their larvicidal activities against Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus, and their antimicrobial activities against Enterococcus faecalis, Staphylococcus aureus, and Candida albicans.
Photographs of the plants presented in this work are shown in Figure 1.
The larvicidal activities of M. leucadendra essential oils are likely due to the high concentrations of α-eudesmol and guaiol, or possibly synergistic effects involving these compounds. Unfortunately, there appear to be no reports on the larvicidal activities of these compounds.

Antimicrobial Activity
The Myrtaceae essential oils were screened for antibacterial activity against Enterococcus faecalis (ATCC 29912) and Staphylococcus aureus (ATCC 25923), and for antifungal activity against Candida albicans (ATCC 10231). The antimicrobial activities are summarized in Table 7. The leaf essential oils of B. frutescens and C. citrinus both showed excellent anti-Candida activity, with minimum inhibitory concentration (MIC) values of 16 µg/mL. van Zyl and co-workers have screened several monoterpenoids against C. albicans, and many of the major components that were found in B. frutescens and C. citrinus leaf essential oils did show notable activities, including α-pinene (MIC 12.0 µg/mL), β-pinene (MIC 1.0 µg/mL), limonene (MIC 10.0 µg/mL), and γ-terpinene (MIC 6.0 µg/mL) [59]. 1,8-Cineole and α-terpineol are relatively inactive against C. albicans, however [60,61]. A perusal of the literature reveals a broad spectrum of reported antimicrobial activities for terpenoid constituents against E. faecalis, S. aureus, and C. albicans (Table 8). There are several potential reasons for the apparent discrepancies, including variation in antimicrobial assay protocols, different susceptibilities of different strains of a particular microorganism, mathematical errors in calculating dilutions and MIC values.

Plant Collection
Plant materials were collected from wild-growing plants in the Hoa Vang and Hoa Khanh districts of Da Nang city. The plants were identified by Do Ngoc Dai. In each case, the fresh plant material was chopped, and 2.0 kg was subjected to hydrodistillation using a Clevenger-type apparatus (Table 9).

Gas Chromatographic -Mass Spectral Analysis
Each of the essential oils was analyzed by gas chromatography-mass spectrometry (GC-MS), as previously reported [81], using a Shimadzu GCMS-QP2010 Ultra, fitted with a ZB-5 column. Identification of the oil components was based on their retention indices determined by reference to a homologous series of n-alkanes, and by comparison of their mass spectral fragmentation patterns with those in the NIST [21] and FFSNC [22] databases and our own Sat-Set library [23].

Mosquito Larvicidal Assays
Mosquito colonies of Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus were obtained and maintained as previously described [82].
Larvicidal activities of the essential oils were evaluated according to the protocol of Liu and co-workers [83] with slight modifications. For each assay, 150 mL of water containing 20 fourth-instar mosquito larvae was placed into 250-mL beakers and aliquots of the essential oils dissolved in EtOH (1% stock solution) were then added. A set of controls using EtOH only (negative control) and permethrin (positive control) were included for comparison. Mortality was recorded after 24 h and after 48 h of exposure, during which no nutritional supplement was added. The experiments were carried out at 25 ± 2 • C. Each test was conducted in quadruplicate with five concentrations (100, 50, 25, 12.5 and 6 µg/mL). The data obtained were subjected to log-probit analysis [84] to obtain LC 50 values, LC 90 values and 95% confidence limits using Minitab ® 19 (Minitab, LLC, State College, PA, USA).

Antimicrobial Screening
The antimicrobial activity of the essential oils was evaluated using two bacteria (Enterococcus faecalis, ATCC 299212, and Staphylococcus aureus, ATCC 25923) and one yeast (Candida albicans, ATCC 10231) using the microdilution broth susceptibility assay, as previously reported [82]. Stock solutions of the each of the essential oils were prepared in dimethylsulfoxide. Dilution series were prepared from 16,384 to 2 µg/mL (2 14 , 2 13 , 2 12 , 2 11 , 2 10 , 2 9 , 2 7 , 2 5 , 2 3 and 2 1 µg/mL) in sterile distilled water in micro-test tubes from where they were transferred to the 96-well microtiter plates for the assays.

Agglomerative Hierarchical Cluster Analysis
The essential oil compositions from this work and from the published literature were treated as operational taxonomic units (OTUs). The percentage composition of the major components of the essential oils was used to determine the chemical relationship between the various essential oil samples by agglomerative hierarchical cluster (AHC) analysis, using the XLSTAT software, version 2018.1.1.6097 (Addinsoft™, Paris, France). Euclidean distance was used to measure dissimilarity, and Ward's method was used for cluster definition.

Conclusions
Essential oils derived from Baeckea frutescens, Callistemon citrinus, Melaleuca leucadendra, and Syzygium nervosum have shown larvicidal activities against the mosquito species tested. In most cases, the larvicidal activities cannot be attributed to the major components, and synergistic interactions with minor components are likely responsible. Likewise, all of the Myrtaceae essential oils examined for antimicrobial activity showed promise. Thus, these essential oils may serve as "green" vector control agents and/or complementary antimicrobial agents, as well as providing value-added commodities for harvested timbers (e.g., Melaleuca leucadendra).