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Larvicidal activity of extracts from Ammi visnaga Linn. (Apiaceae) seeds against Culex quinquefasciatus Say. (Diptera: Culicidae)
Experimental Parasitology 165 (2016) 51e57
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Full length article
Larvicidal activity of extracts from Ammi visnaga Linn. (Apiaceae) seeds against Culex quinquefasciatus Say. (Diptera: Culicidae) b, Jan Tríska b Roman Pavela a, *, Nade zda Vrchotova a b
Crop Research Institute, Drnovska 507, 161 06 Prague 6, Ruzyne, Czech Republic 31, Cesk Global Change Research Centre Academy of Science Czech Republic, Branisovska e Bud ejovice, Czech Republic
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The furanochromene khellin and visnagin as active substances suitable for the development of botanical insecticides. Larvicidal activity the extracts from Ammi visnaga seeds against Culex quinquefasciatus. The effect of the short action of the extract, visnagin and khellin on C. quinquefasciatus larvae. The effect of the lethal concentrations on C. quinquefasciatus larval mortality. The exposure time needed to achieve 30%, 50% or 90% mortality of the 3rd instar larvae of C. quinquefasciatus was determined.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 April 2015 Received in revised form 29 September 2015 Accepted 14 March 2016 Available online 16 March 2016
Efficacies of the Ammi visnaga seeds extract and a majority of substances on larval Culex quinquefasciatus mortality in various development stages including pupae were studied. The effect of exposure time on larval mortality was also studied. The effect of sublethal concentrations or short exposure times on further larval development and subsequent fecundity in adults were studied as well. Lethal doses of the extract were estimated for the 2nd, 3rd and 4th instar of C. quinquefasciatus (LC50 for 18, 23 and 180 mg L1, respectively). The majority of furanochromenes, khellin and visnagin, were identified by analysing the extract. Khellin was significantly more effective compared to visnagin, whose LC50 was estimated at 8, 10 and 41 mg L1 for the 2nd, 3rd and 4th instar larvae. Khellin showed very fast efficacy on mortality for the 3rd instar larvae in a concentration of 100 mg L1. Fifty percent mortality was determined 30 min after application, a time which was considerably shorter compared to the extract (113 min) or visnagin (169 min). The effect of the application of lethal concentrations on C. quinquefasciatus larval mortality was studied. The least number of adults were hatched after application of the extract and khellin (41.8% and 37.9%, respectively), less than after visnagin application (46.7%) or in the control (94.2%). LC50 application caused lower fecundity in the hatched adults, lower hatchability of the eggs, and also very low natality, more than 77% lower for khellin compared to the control.
Keywords: Botanical larvicide Furanochromenes Visnagin Khellin Sublethal effect Mosquito
* Corresponding author. E-mail address: [email protected] (R. Pavela). http://dx.doi.org/10.1016/j.exppara.2016.03.016 0014-4894/Crown Copyright © 2016 Published by Elsevier Inc. All rights reserved.
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A short exposure, corresponding to our estimated LT30, caused no significant acute toxicity in the larvae (until 24 h) for the extract or visnagin (4.3% and 11.5%, respectively); however, 18 min of action from khellin caused a 54.3% mortality rate of the larvae within 24 h. Crown Copyright © 2016 Published by Elsevier Inc. All rights reserved.
1. Introduction Mosquitoes are cosmopolitan and common as well as transmit a variety of diseases, while also being a nuisance. The Culex quinquefasciatus Say (Diptera: Culicidae) causes angioedema, a cutaneous allergy in humans which is also responsible for the transmission of some zoonotic pathogens (Brown, 1986; WHO, 1992). For example, C. quinquefasciatus is the dominant vector in the transmission of a number of arboviruses, or lymphatic filariasis, a disease caused by threadlike parasitic worms. Filariasis, which has infected some 120 million people worldwide, can lead to genital damage and elephantiasis (WHO, 1992; WHO, 2010). The global mosquito control strategy aims at protecting individuals and communities using long-lasting impregnated nets, indoor-residual spraying in addition to prompt and effective clinical treatment (Fillinger and Lindsay, 2011). Since long-lasting impregnated nets and indoor-residual spraying are directed against the adult vector population that enters houses, further suppression of transmission could be achieved by targeting the aquatic stages by reducing vector larval habitats, thus attacking both outdoor and indoor biting vectors. This may be particularly important in areas targeted for elimination where vector foci or 'hot spots' persist (Bejon et al., 2010; Cohen et al., 2010). The historical literature and more recent reviews of this approach show that anti-larval mosquito control measures are powerful tools against vectors. Significantly, larval source management contributed to all successful eradication efforts and successful vector-control programmes worldwide. Mosquito larvae are relatively immobile and often readily accessible. By targeting the larval stages, mosquitoes are killed ‘wholesale’ before they disperse to human habitations. Mosquito larvae, unlike adults, cannot change their habitat to avoid control activities (Fillinger and Lindsay, 2011). However, the use of insecticides faces several serious problems today. In addition to the negative effects of synthetic insecticides on the environment and non-target organisms, including man (Hodgson and Levi, 1996; Paoletti and Pimentel, 2000; Roberts and Karr, 2012), the development of resistant mosquito populations in particular is one of the most serious problems (WHO, 2012a). These problems have become the main impetus for an expeditious search for new alternatives, which would be acceptable for both the environment and health, for protection against insects. Among the existing alternative strategies aimed at decreasing vector populations, the use of pesticides based on plant extracts is currently one of the most promising. The use of botanical insecticides is a plant and vector protection alternative, generally considered safe for the environment and health (Copping and Menn, 2000; Pavela, 2014; Senthil-Nathan, 2015). Therefore, significant efforts are currently being devoted to the search for new, highly efficient plant extracts which would be suitable for the development of botanical insecticides (Miresmailli and Isman, 2014). Moreover, botanical insecticides usually contain a mixture of several active substances which exert different mechanisms of action as a rule, and thus may be able to effectively prevent the
emergence of resistant insect populations (Rattan, 2010; RegnaultRoger et al., 2012). Considerable efforts have been devoted in the past few decades to research on the biological activity of plant extracts against various insects (Pavela, 2011b; Attia et al., 2013; Senthil-Nathan, 2013; Pavela, 2013) including mosquitoes (Pavela, 2009a,b; Dias and Moraes, 2014). This research helps to find plant species that can provide active substances with a novel mechanism of effect, and are also associated with minimal health-related and environmental risks. Among these extracts, several can also be used for the development and production of botanical larvicides against important vectors (Sukumar et al., 1991; Howard et al., 2011). By comparing lethal doses of extracts obtained from plants of the Eurasian Region, several species were suitable selections for the development of botanical insecticides in one of our previous studies (Pavela, 2008, 2009a,b). The selected plants also included Ammi visnaga L. (Apiaceae), whose methanol extracts showed excellent insecticidal efficacy against C. quinquefasciatus larvae in the tests (Pavela, 2008). A. visnaga is one of the most important medicinal plants. It was used in ancient Egypt as a herbal remedy for renal colic. A. visnaga fruit preparations, such as tea prepared from crushed or powdered seeds, have traditionally been used in the Middle East to ease urinary tract pain associated with kidney stones and to promote stone passage (Gunaydin and Beyazit, 2004). Khellin has been used for the treatment of urologic, dermatologic, and respiratory symptoms. It is used in the management of bronchial asthma and angina pectoris. The plant also possesses antimicrobial activity and inhibits certain mutagens (Vedaldi et al., 1988). Taking into account that the extracts, including visnagin and khellin, have been used in popular medicine, botanical insecticides based on these substances can be considered safe for ones health. Therefore, the purpose of this study was to find more details on the biological efficacy of extracts from A. visnaga seeds and appropriate majority of substances against C. quinquefasciatus larvae. In particular: i. Efficacy of the extract and majority of substances on larval mortality at various development stages including pupae; ii. The effect of exposure time on larval mortality; and iii. The effect of sublethal concentrations or short exposure times on further larval development and subsequent fecundity in adults. The results of our work are important for estimating a possible mechanism of action and application method for A. visnaga extract as a potential larvicide. 2. Materials and methods 2.1. 1. Plant extract and analysis The seeds of A. visnaga were harvested in the autumn of 2012 from the CRI - Praha field and dried in the institute's laboratory at room temperature. Subsequently, the seeds were ground and extracted using pure methanol (MeOH) at the ratio of 1:10 (seeds: MeOH). The extraction was done for 48 h while stirring the solution from time to time; the extract was then filtered and thickened in vacuo at an operating temperature of 40 C. The obtained extract was stored in the dark, at 3 C, until the experiments. An extract
R. Pavela et al. / Experimental Parasitology 165 (2016) 51e57
sample as well as parts of the harvested plants and seeds have been stored at the RCI laboratory, Voucher No. 12009. The extract from A. visnaga was analysed by HPLC (Hewlett Packard Ti-1050) with a DAD detector (Agilent G1315B). Separation was performed on the reversed phase column (Phenomenex Luna C18, 150 2 mm, 3um) in gradient water-acetonitrile-methanolphosphoric acid at 35 C. Mobile phase A: 5% acetonitrile þ0.1% o-phosphoric acid, mobile phase B: 80% acetonitrile þ0.1% o-phosphoric acid and mobile phase C: 95% methanol. Gradient: 0. min (70% A, 20% B, 10% C), 25. min (70% A, 20% B, 10% C), 30. min (10% A, 80% B, 10% C), 35. min (90% B, 10% C), 40. min (90% B, 10% C). Flow was 0.25 ml/min. Khellin and visnagin were detected at 220 nm. Quantities of substances were calculated according to the calibration curves corresponding standards (Sigma Aldrich, Czech Republic, purity >99%). 2.2. Mosquitoes The test organism, C. quinquefasciatus, was reared in the laboratory, Secondary Plant Metabolites in Crop Protection, Crop Research Institute (Czech Republic). They were maintained at 25 ± 2 C and 75e85% relative humidity under 14:10 light and dark cycles. Pupae were transferred from the trays to a plastic cup (10 5 3 cm) containing tap water and were maintained in an insectary (45 45 40 cm) where adults emerged. Adults were maintained in plastic cages and were continuously provided with a 10% sucrose solution in a jar with a cotton wick. On day 5, the adults were given a blood meal from a chick placed in resting cages overnight for blood feeding by females. A glass petri dish with 50 ml of tap water lined with filter paper was kept inside the cage for oviposition (Pavela, 2011a). 2.3. Experimental 2.3.1. Larvicidal activity Mosquito larvicidal assays were carried out according to WHO (1996) standard procedures, with slight modifications (Pavela, 2008). The extract or furanochromenes were diluted in dimethyl sulphoxide (DMSO) to prepare a serial dilution of the test dosage. The furanochromenes visnagin and khellin, purity >98%, were obtained from Sigma Aldrich (Czech Republic) and used in the tests. Early 2nd, 3rd or 4th instar larvae of C. quinquefasciatus were selected and transferred in 224 ml of distilled water. For experimental treatment, 1 ml of serial dilutions was added to 224 ml of distilled water in a 500-ml glass bowl and shaken lightly to ensure a homogenous test solution. The selected larvae were transferred in distilled water into a bowl of prepared test solution with a final surface area of 125 cm2 (25 larvae/beaker). Four replicates were run simultaneously with at least ten dosages (from 2 to 200 mg L1). The assays were placed in a growth chamber (L16:D9, 25 ± 1 C). Mortality was determined after 24 h of exposure, during a time which no food was offered to the larvae. 2.3.2. Time needed for mortality The extract, visnagin or khellin, was applied in a concentration of 100 mg L1 to the 3rd instar larvae of C. quinquefasciatus in order to determine the activity rate of the extract and appropriate furanochromenes on larval mortality. The fundamental methodology of establishing the experiments and experimental conditions was the same as described in the previous chapter, 2.3.1. Larvicidal activity, with the only difference being that larval mortality was recorded in a time series as follows:
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Mortality was recorded every 5 min during the first 30 min, and then at intervals of 10e15 min from 30 min to 200 min of the experiment. Any larvae that showed no signs of movement after mechanical stimulation were considered dead. In order to estimate the activity rate, 7 different time intervals were used in which mortality was determined in the range from 10% to 95%. The experiment was replicated 3 times. The assays were placed in a growth chamber (L16:D9, 25 ± 1 C). 2.3.3. Effect of lethal concentrations on larval development and fertility of surviving adults At the beginning of the 3rd instar, the larvae were put into a plastic container (20 20 20 cm) with 3 l of drinking water. Upon acclimatization (after approximately 1 h), a dose of essential oil was mixed into the water, corresponding to the calculated concentration LD50 (for the extract, visnagin and khellin, 23, 26, and 10 mg L1, respectively, Table 1). The extract or furanochromenes were emulsified using DMSO; water with an adequate DMSO content was used for the control larvae. There were 100 larvae in each container at the beginning; the assay was repeated three times. After 24 h of exposure, the larvae were transferred into clean water, where they were left until the incubation of adults. Larval mortality was determined after 24 and 48 h of exposure; total mortality, percentage of incubated adults, and their sex were determined. The larvae were fed with dog biscuits and yeast powder at a 3:1 ratio. Always 20 females and 20 males were randomly selected from incubated adults in order to determine the effect of LC50 on fecundity, fertility and natality. The adults of both sexes were placed together in breeding cages (25 25 30 cm). The adults were fed on a sugar solution, and blood feeding was provided after 7 days. A bowl with water (10 cm in diameter) was placed in the cage for oviposition. The number of oviposited eggs was determined every day (using a microscope) and a defined number of eggs was always left in order to determine their hatchability. The obtained data was used to calculate the following indicators: Fecundity ¼ E/F where E is the sum of oviposited eggs and F is the number of females that entered the experiments; Fertility (%) ¼ (H/ O)*100 where H is the number of hatched larvae and O is the total number of eggs; Natality (%) ¼ (O FEM)/L where O is the mean count of hatched larvae per female, FEM is the number of hatched females, and L is the number of larvae that entered the experiment (Pavela, 2007). The experiment was repeated 4 times. The assays were placed in a growth chamber (L16:D9, 25 ± 1 C). 2.3.4. Effect of short-term exposure on subsequent larval development and fertility of surviving adults The experiment described below was undertaken in order to determine the short-term activity of the extract or appropriate furanochromenes on larval development and subsequent fertility of surviving adults. The 3rd instar larvae of C. quinquefasciatus were exposed to a concentration of 100 mg L1 for a time period corresponding to the estimated LT30 (i.e., 75, 43 and 18 min for the extract, visnagin and khellin, respectively, see Table 2). The methodology of establishing the experiment was identical to that of 2.3.3. Effect of lethal concentrations on larval development and fertility of surviving adults, with the only difference being that after the exposure period, the larvae were transferred to clean water. The experiment was repeated 4 times. The assays were placed in a growth chamber (L16:D9, 25 ± 1 C). 2.3.5. Statistical analysis The mortality was corrected by Abbott's formula (Abbott, 1925) and an LC50, 99 and LT30, 50, 90 regression equation; a 95% confidence limit was calculated by using probit analysis (Finney, 1971).
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Table 1 Toxicity of extract and two furochromones to juvenile developmental stages of Culex quinquefasciatus during a 24 h of exposure. Extract
2nd instar 3rd instar 4th instar pupae a b
Visnagin
Khellin
LC50 (CI95)a mg.L1
LC99 (CI95)a mg.L1
Chib
LC50 (CI95)a mg.L1
LC99 (CI95)a mg.L1
Chib
LC50 (CI95)a mg.L1
LC99 (CI95)a mg.L1
Chib
18 (13e22) 23 (21e35) 180 (158e230) >200
42 (36e58) 58 (45e67) >200 >200
2.154 2.527 3.456
22 (20e25) 26 (24e28) 52 (48e59) >200
40 (35e49) 41 (37e46) 79 (65e82) >200
2.992 4.715 1.557
8 (5e9) 10 (7e12) 41 (39e49) >200
26 (22e32) 23 (21e28) 65 (59e72) >200
0.127 0.128 1.258
Ninety-five percent confidence intervals (CI), essential oils activity is considered significantly different when the 95% CI fail to overlap. Chi-square value, significant at P < 0.05 level.
Table 2 Time required to achieve 30%, 50% or 90% mortality of third instars of Culex quinquefasciatus.
Extract Visnagin Khellin
LT30 (CI95)a
LT50 (CI95)a
LT90 (CI95)a
Chib
75 (58e85) 43 (32e51) 18 (12e21)
113 (106e120) 169 (159e181) 30 (27e32)
184 (170e204) 276 (247e324) 51 (46e58)
2.929 1.266 4.388
Lethal time was calculated as time (in minutes) to indigent of 30%, 50% or 90% mortality of third instars of C. quinquefasciatus. Larvae were exposed to doses corresponding to the estimated LC99 (58, 41 and 23 mg L1 for extract, visnagin and khellin, respectively). a Ninety-five percent confidence intervals (CI), essential oils activity is considered significantly different when the 95% CI fail to overlap. b Chi-square value, significant at P < 0.05 level.
The percentage of mortality, emergence of adults and fecundity indicators were determined and transformed to arcsine square root values for analysis of variance.(ANOVA) Tukey's test (P < 0.05) was used to analyse significant differences against mosquitoes between the test extract and furanochromenes. 3. Results An extract of honey-like colour was obtained by extraction of A. visnaga seeds, with a yield of 4.87%. By analysing the extract, two majority substances belonging to the group of furanochromenes were determined: khellin and visnagin (Fig. 1), whose content was determined as 361.9 and 235.7 mg g1, respectively. In general, these two majority substances represented almost 60% of the extract content. The effect of the extract and the two determined furanochromenes on larval and pupal mortality is shown in Table 1. A significant difference was found in extract efficacies for individual development stages. While no significant difference between larvicidal efficacy of the extract was found between the 2nd and 3rd instars (LC50 ¼ 18 and 23 mg L1; LC90 ¼ 42 and 58 mg L1; respectively), the 4th instar larvae were significantly less sensitive (LC50 ¼ 180 mg L1; LC90 > 200 mg L1), and in the pupal stage, the extract caused no significant mortality (LC50 > 200 mg L1). Based on comparing the LC50 values of the furanochromenes themselves, khellin was found to be significantly more efficient against the 2nd and 3rd instar larvae of C. quinquefasciatus (LC50 ¼ 8 and 10 mg L1 for the 2nd and 3rd instar, respectively) compared to visnagin (LC50 ¼ 22 and 26 mg L1 for the 2nd and 3rd instar, respectively). In the last instar, the significance of this phenomenon decreased (P < 0.05), and none of the furanochromenes caused mortality in the pupal stage. The exposure time needed to achieve 30%, 50% or 90% mortality of the 3rd instar larvae of C. quinquefasciatus is shown in Table 2. The extract, visnagin and khellin were applied in uniform concentrations of 100 mg L1 to make sure that the time would not be affected by varying dosages. Khellin provided the fastest effect,
causing 50% and 90% mortality after 30 and 51 min, respectively, and thus in a significantly shorter time than the extract and visnagin. However, the extract provided a significantly faster effect (LT50 ¼ 113 min; LT90 ¼ 184) compared to visnagin (LT50 ¼ 169 min; LT90 ¼ 276 min). The effect of the application in lethal concentrations on C. quinquefasciatus larval mortality is presented in Table 3. Mortality 24 h after application reached approximately 50%, as expected, and kept rising only slightly during the course of larval development (by approximately 2e9%, depending on the variant). In general, significantly fewer adults hatched in all variants compared to the control. The least number of adults were hatched after the application of the extract and khellin (41.8% and 37.9%, respectively), thus less than after visnagin application (46.7%) or in the control (94.2%). LC50 application caused lower fecundity in the hatched adults (Table 3), lower hatchability of the eggs, and consequently very low natality, more than 77% lower for khellin compared to the control. The effect of the short action of the extract, visnagin and khellin on C. quinquefasciatus larvae is presented in Table 4. A short exposure, corresponding to our estimated LT30, caused no significant acute toxicity in the larvae (until 24 h) for the extract or visnagin (4.3% and 11.5%, respectively); however, 18 min of action from khellin caused a 54.3% mortality rate of the larvae within 24 h. The highest total larval mortality was recorded for khellin (65.3%) and the lowest for the extract (18.7%). Although the adults who hatched from surviving pupae showed lower fecundity after application of the extract and visnagin (Table 4), a significantly lower number of eggs per female (P > 0.05) was found only for visnagin. The effect of the extract, visnagin and khellin led to significantly lower total natality, lower by 67.6%, 53.3% and 34.8%, respectively, compared to the control. 4. Discussion In a previous study, we determined that the methanol extract from A. visnaga seeds had an excellent effect against C. quinquefasciatus larvae (Pavela, 2008). From this point of view, our current study confirms the results. However, the efficacy of the extract was newly found to significantly decrease in the last development instar of the larvae, and no effect was exerted on mortality in the pupal stage. This indicates that practical application of the extract should be aimed at early development stages of the mosquitoes. No studies of the efficacy of furanochromenes such as visnagin and khellin on mosquito larval mortality have been published until now and therefore we cannot safely compare our results with those of other authors. However, the acaricidal efficacy of an extract from A. visnaga has been known (Pavela, 2015a). The adult mortality of Tetranychus urticae was studied both for the extract from A. visnaga seeds and for visnagin and khellin. Although both of these furanochromenes were found to have a significant acaricidal effect, only visnagin showed an effect on acute toxicity (LD50 for
R. Pavela et al. / Experimental Parasitology 165 (2016) 51e57
55
DAD1 B, Sig=246,4 Ref=450,40 (ZK002934.D) mAU
175 OCH 3 O
O
150
CH3
visnagin
khellin
125
O
O
OCH 3
CH3
O
100 OCH 3
O
75
50
25
0
-25
0
5
10
15
20
25
30
35
min
Fig. 1. HPLC chromatogram of the methanolic extract of the Ammi visnaga seeds.
24 h ¼ 25 mg cm2). However, both of these substances showed a long persistence period and significant efficacy in terms of chronic toxicity. Moreover, they exhibited a very strong ovicidal efficacy (LD50 ¼ 0.5 and 1.8 mg cm2, for visnagin and khellin, respectively). The determined furanochromenes e visnagin and khellin e represented almost 60% of the extract content, and in the tests it was also demonstrated that they were responsible for the larvicidal efficacy of the extract. As for the biological efficacy of both furanochromenes, khellin was more effective than visnagin; although this significant difference declined in the last development stage, both furanochromenes also retained good efficacy against the 4th instar larvae. The furanochromenes had no effect on pupal mortality. On the contrary, as determined for the acaricidal efficacy (Pavela, 2015a), visnagin provided a much higher efficacy against all stages of T. urticae. In addition, based on our observations, the extract from A. visnaga including both of the tested substances is
not efficient against some insects (such as aphids), and it does not cause any acute or chronic toxicity (not yet published results). This would indicate that botanical insecticides based on the extract from A. visnaga are selective, which is important in terms of the environmental safety of these potential products. Comparing the lethal doses determined by us with the results of other authors, we can note that both the extract and the two active substances showed excellent biological efficacy. For the 3rd instar of C. quinquefasciatus larvae, we determined LC50 values of 23, 26 and 10 ppm (for the extract, Visnagin and Khellin, respectively). Comparing our LC50 values with the lethal concentrations of essential oils (EOs), for example, which are generally considered to have great potential for the development of botanical insecticides including larvicides (Dias and Moraes, 2014), we can note that the extract from A. visnaga shows an efficacy in terms of acute toxicity similar to the most efficient EOs. As was found (Pavela, 2015b) based on a recent evaluation of the larvicidal efficacy of EOs, only
Table 3 Effect of short time exposition of extract and furochromones on emergence and fecundity of Culex quinquefasciatus adults that survived after treatment in the larvae stage. Larval mortality (%)*
Extract Visnagin Khellin Control F, P**
Emergence of adults (%)*
Fecundity indicators*
At 24 h
At 48 h
Total
Female
Male
Total
Fecundity (No. Eggs/ female)
Fertility (% egg hatchability)
Natality (Number of larvae/ larvae)
Inhibition of natality (%)
4.3 ± 0.3b 11.5 ± 4.7c 54.3 ± 5.1d 0.0 ± 0.0a 378.27, 0.001
4.3 ± 0.3b 11.8 ± 5.1c 58.0 ± 4.4d 0.0 ± 0.0a 156.11, 0.001
18.7 ± 4.9b 27.7 ± 5.6c 65.3 ± 6.0d 2.1 ± 0.7a 201.17, 0.001
36.1 ± 2.3b 35.7 ± 5.0b 17.1 ± 1.8a 48.1 ± 0.9c 229.79, 0.001
41.0 ± 2.8c 33.8 ± 2.0b 14.5 ± 4.3a 47.5 ± 0.5d 213.29, 0.001
77.1 ± 5.1c 69.5 ± 6.0b 32.0 ± 6.1a 96.3 ± 0.4d 516.64, 0.001
145.4 ± 15.1ab 135.8 ± 21.2a 182.4 ± 11.3b 179.3 ± 21.6b 3.96, 0.035
92.6 ± 5.9 82.8 ± 20.1 87.0 ± 17.2 98.5 ± 1.3 0.12, 0.896
55.4 ± 4.5b 39.7 ± 5.6ab 27.6 ± 2.9a 85.1 ± 1.6c 39.12, 0.001
34.8 ± 4.7a 53.3 ± 5.8ab 67.6 ± 3.1b 42.05, 0.003
The 3rd instar larvae of C. quinquefasciatus were exposed to concentrations corresponding to the estimated LC99 (58, 41 and 23 mg L1 for extract, visnagin and khellin, respectively) for the time (75, 43 and 17 min for extract, visnagin and khellin, respectively) required to achieve 30% mortality of the larvae. * Mean % (±S.E.) within a column follower by the same letter do not differ significantly according to the least significant difference (LSD) test at P < 0.05 (% ¼ arc sine transformed data). ** ANOVA parameters - F-value, P-significantly level.
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Table 4 Emergence and fecundity of Culex quinquefasciatus adults that survived sublethal concentration of extract and furochromones after treatment in the larvae stage. Mortality (%)*
Extract Visnagin Khellin Control F, P**
Emergence of adults (%)*
Fecundity indicators
Total
Fecundity (no. Eggs/female)
Fertility (% egg hatchability)
Natality (number of larvae/larvae)
41.8 ± 3.1ab 46.7 ± 4.2b 37.9 ± 2.2a 94.2 ± 5.6c 178.44, 0.001
133.1 ± 25.2a 143.2 ± 21.5ab 143.6 ± 12.9a 169.9 ± 11.3b 3.68, 0.026
84.1 ± 3.2a 84.9 ± 3.6a 92.8 ± 9.2ab 98.3 ± 5.3b 4.77, 0.007
23.9 28.4 17.7 79.7 69.3
At 24 h
At 48 h
Total
Female
Male
52.6 ± 7.2b 51.1 ± 9.9b 52.6 ± 7.1b 0.0 ± 0.0a 155.59, 0.001
54.9 ± 5.3bc 51.1 ± 9.9b 58.6 ± 3.9c 0.0 ± 0.0a 346.96, 0.001
57.8 ± 4.9bc 53.1 ± 8.3b 61.63 ± 6.1c 4.4 ± 2.3a 185.66, 0.001
18.5 ± 2.5ab 23.2 ± 6.2b 13.3 ± 2.3a 51.3 ± 3.9c 44.3, 0.001
23.4 ± 23.4 ± 24.7 ± 42.8 ± 10.96, 0.001
3.8a 3.5a 2.9a 5.2c
*
± 4.2ab ± 1.7b ± 1.7a ± 5.3c 0.001
Inhibition of natality (%) 70.1 ± 5.3ab 64.4 ± 1.9a 77.7 ± 2.1b 5.37, 0.008
The 3rd instar larvae of C. quinquefasciatus were 24 h exposed to concentrations corresponding to the estimated LD50 (23, 26 and 10 mg L1 for extract, visnagin and khellin, respectively) for 24 the time (75, 43 and 17 min for extract, visnagin and khellin, respectively). * Mean % (±S.E.) within a column follower by the same letter do not differ significantly according to the least significant difference (LSD) test at P < 0.05 (% ¼ arc sine transformed data).**ANOVA parameters - F-value, P-significantly level.
122 plant species fulfilled two essential conditions: (i) LC50 100 ppm; and (ii) their chemical composition had to be known. Considering the above-estimated LC50 value as the main criterion of efficacy, 77 EOs showed LC50 < 50 ppm. Some of these efficient EOs were obtained from aromatic plants also grown commercially on relatively large areas, with a good technology of cultivation (e.g., Pimpinella anisum, Coriandrum sativum, Foeniculum vulgare, Mentha longifolia, Ocimum basilicum, Thymus spp., Eucalyptus spp., Piper spp., etc.). Only seven plants (Blumea densiflora, Auxemma glazioviana, Callitris glaucophylla, Cinnamomum microphyllum, Cinnamomum mollissimum, Cinnamomum rhyncophyllum, Zanthoxylum oxyphyllum) can be considered significantly most efficient, given that LC50 < 10 ppm has been estimated for their EOs. These EOs contained less common substances, predominantly from the group of sesquiterpenes, aromatic acids and ketones. When larvicides are applied onto a not quite stationary water surface, the applied dose may be gradually diluted by water flowing in or out, and thus their efficacy may be reduced. For this reason, the exposure time of the active substance, i.e., the time necessary to ensure the required biological efficacy, should be known. We therefore tested the activity rate for the concentration 100 mg L1, which is considered relatively acceptable for practical application (WHO, 2012b; Dias and Moraes, 2014; Bezerra-Silva et al., 2015). The extract from A. visnaga provided a relatively rapid effect, achieving 50% or 90% larval mortality within approximately 2 or 3 h after application, respectively. As for the tested furanochromenes, the most rapid effect was provided by khellin alone, which led to 90% mortality after less than 1 h from application. However, even a short exposure time or the application of lethal concentrations of the extract or its active substances showed a significant effect on overall mosquito natality in our tests. As found previously, some plant substances can lead to a significant reduction in fecundity and thus also overall natality in some vectors, even in lethal or sublethal doses or concentrations; for example, this is also the case for essential oils from Thymus vulgaris (Pavela, 2007). Our results indicate that the mechanism of effect of the A. visnaga extract is selective in terms of development stages of the mosquito, given that acute toxicity was manifested only in larval stages, while the application of the extract or furanochromenes had no effect on the pupae (not even long-term exposure to relatively high concentrations of 200 mg L1, as observed in our orientation experiments not yet published). This extract may also provide interspecies selectivity as, for example, no effect on mortality or other development characteristics in the larvae of Spodoptera littoralis (observations not yet published) has been seen in our tests. These observations of ours may indicate selectivity of the extract with respect to non-target organisms. However, further experiments will be needed to confirm this hypothesis. In conclusion, we can recommend the extract from A. visnaga
seeds or mixture of the furanochromene khellin and visnagin as active substances suitable for the development of botanical insecticides. We can recommend application of these potential botanical insecticides as larvicidal products against mosquito larvae. However, further tests with formulations of these products will be needed; such tests should be carried out at suitable localities and should include observation of their effects on non-target organisms. Acknowledgements Financial support for this work was provided by the Technology Agency of the CR (Project no. TA04020103) and CzechGlobe (Reg.N. CZ.1.05/1.1.00/02.0073). References Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 8, 265e267. Attia, S., Grissa, K.L., Lognay, G., Bitume, E., Hance, T., Mailleux, A.C., 2013. A review of the major biological approaches to control the worldwide pest Tetranychus urticae (Acari: Tetranychidae) with special reference to natural pesticides. J. Pest Sci. 86, 361e386. Bejon, P., Williams, T.N., Liljander, A., Noor, A.M., Wambua, J., Ogada, E., Olotu, A., Osier, F.H.A., Hay, S.I., Farnert, A., Marsh, K., 2010. Stable and unstable malaria hotspots in longitudinal cohort studies in Kenya. PLoS Med. 7 (7), e1000304. http://dx.doi.org/10.1371/journal.pmed.1000304. Bezerra-Silva, P.C., Santos, J.C., Santos, G.N.K., Dutra, K.A., Santana, A.L.B.D., Maranh~ ao, C.A., Nascimento, M.S., Navarro, D.M.A.F., Bieber, L.W., 2015. Extract of Bowdichia virgilioides and maackiain as larvicidal agent against Aedes aegypti mosquito. Exp. Parasitol. 153, 160e164. Brown, A.W.A., 1986. Insecticide resistance in mosquitoes: pragmatic review. J. Am. Mosq. Control. Assoc. 2, 123e140. Cohen, J.M., Moonen, B., Snow, R.W., Smith, D.L., 2010. How absolute is zero? An evaluation of historical and current definitions of malaria elimination. Malar. J. 9 (1), 213. Copping, L.G., Menn, J.J., 2000. Biopesticides: a review of their action, applications and efficacy. Pest Manag. Sci. 56, 651e676. Dias, N., Moraes, D.F.C., 2014. Essential oils and their compounds as Aedes aegypti L. (Diptera: Culicidae) larvicides: review. Parasitol. Res. 113, 565e592. Finney, D.J., 1971. Probit Analysis. Cambridge University Press, London. Fillinger, U., Lindsay, S.W., 2011. Larval source management for malaria control in Africa: myths and reality. Fill. Lind. Malar. J. 10, 353. Gunaydin, K., Beyazit, N., 2004. The chemical investigations on the ripe fruits of Ammi visnaga (Lam.) Lamarck growing in Turkey. Nat. Prod. Res. 18, 169e175. Hodgson, E., Levi, P.E., 1996. Pesticides: an important but underused model for the environmental health sciences. Environ. Health Persp 104, 97e106. Howard, A.F.V., Adongo, E.A., Vulule, J., Githure, J., 2011. Effects of a botanical larvicide derived from Azadirachta indica (the neem tree) on oviposition behaviour in Anopheles gambiae s.s. mosquitoes. J. Med. Plant. Res. 5, 1948e1954. Miresmailli, S., Isman, M.B., 2014. Botanical insecticides inspired by planteherbivore chemical interactions. Trend Pl. Sci. 19, 29e35. Paoletti, M.G., Pimentel, D., 2000. Environmental risks of pesticides versus genetic engineering for agricultural pest control. J. Agric. Environ. Ethic 12, 279e303. Pavela, R., 2007. Lethal and sublethal effects of thyme oil (Thymus vulgaris L.) on the house fly (Musca domestica Lin.). J. Essent. Oil. Bear. Pl. 10, 346e356. Pavela, R., 2008. Larvicidal effects of various Euro-Asiatic plants against Culex quinquefasciatus Say larvae (Diptera: Culicidae). Parasit. Res. 102, 555e559.
R. Pavela et al. / Experimental Parasitology 165 (2016) 51e57 Pavela, R., 2009a. Larvicidal effects of some Euro-Asiatic plants against Culex quinquefasciatus Say larvae (Diptera: Culicidae). Parasit. Res. 105, 887e892. Pavela, R., 2009b. Larvicidal property of essential oils against Culex quinquefasciatus Say (Diptera: Culicidae). Ind. Crop Prod. 30, 311e315. Pavela, R., 2011a. Insecticidal properties of phenols on Culex quinquefasciatus Say and Musca domestica L. Parasit. Res. 109, 1547e1553. Pavela, R., 2011b. Antifeedant and larvicidal effects of some phenolic components of essential oils last lines of introduction against Spodoptera littoralis (Boisd.). J. Essent. Oil Bear. Plants 14, 266e273. Pavela, R., 2013. Efficacy of naphthoquinones as insecticides against the house fly, Musca domestica L. Ind. Crop. Prod. 43, 745e750. Pavela, R., 2014. Insecticidal properties of Pimpinella anisum essential oils against the Culex quinquefasciatus and the non-target organism Daphnia magna. J. Asia Pacif. Entomol. 17, 287e293. Pavela, R., 2015a. Essential oils for the development of eco-friendly mosquito larvicides: a review. Ind. Crop. Prod. 76, 174e187. Pavela, R., 2015b. Acaricidal properties of extracts and major furanochromenes from the seeds of Ammi visnaga Linn. against Tetranychus urticae Koch. Ind. Crop. Prod. 67, 108e113. Rattan, R.S., 2010. Mechanism of action of insecticidal secondary metabolites of plant origin. Crop Prot. 29, 913e920. Regnault-Roger, C., Vincent, C., Arnason, J.T., 2012. Essential oils in insect control: low-risk products in a high-stakes world. Ann. Rev. Entomol. 57, 405e424. Roberts, J.R., Karr, C.J., 2012. Pesticide exposure in Children. Counc. Environ. Health.
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Pediatr. 130, E1765eE1788. http://dx.doi.org/10.1542/peds.2012-2758. Senthil-Nathan, S., 2013. Physiological and biochemical effect of Neem and other Meliaceae plants secondary metabolites against Lepidopteran insects. Front. Physiol. 4, 359. http://dx.doi.org/10.3389/fphys.2013.00359. Senthil-Nathan, S., 2015. A review of biopesticides and their mode of action against insect pest's. In: Environmental Sustainability- Role of Green Technologies. Springer-Verlag, pp. 49e63. Sukumar, K., Perich, M.J., Boobar, L.R., 1991. Botanical derivatives in mosquito control: a review. J. Am. Mosq. Control. Assoc. 72, 210e237. Vedaldi, S., Caflleri, F., Dall'Acqua, L., Andreassi, L., Bovalini, L., Martelli, P., 1988. Khellin, a naturally occurring furochromone, used for the photochemotherapy of skin diseases: mechanism of action. Farm. Sci. 43, 333e346. WHO, 1992. Vector resistance to insecticides. 15th report of the WHO expert committee on vector biology and control. Tech. Rep. Ser. 818, 1e62. WHO, 1996. Report of the WHO Informal Consultation on the Evaluation and Testing of Insecticides. CTD/WHOPES/IC/96 1:69. WHO, 2010. Global Information System on Alcohol and Health. Geneva. www.who. int/globalatlas/DataQuery/default.asp. WHO, 2012a. Global Plan for Insecticide Resistance Management in Malaria Vectors (GPIRM). http://whqlibdoc.who.int/Publications/2012/9789241564472_eng.pdf (accessed 15.04.2012.). WHO, 2012b. Interim Position Statement. The Role Oflarviciding For Malaria Control In Sub-Saharan Africa. Global Malaria Program, Geneva, Switzerland.
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Full length article
Larvicidal activity of extracts from Ammi visnaga Linn. (Apiaceae) seeds against Culex quinquefasciatus Say. (Diptera: Culicidae) b, Jan Tríska b Roman Pavela a, *, Nade zda Vrchotova a b
Crop Research Institute, Drnovska 507, 161 06 Prague 6, Ruzyne, Czech Republic 31, Cesk Global Change Research Centre Academy of Science Czech Republic, Branisovska e Bud ejovice, Czech Republic
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The furanochromene khellin and visnagin as active substances suitable for the development of botanical insecticides. Larvicidal activity the extracts from Ammi visnaga seeds against Culex quinquefasciatus. The effect of the short action of the extract, visnagin and khellin on C. quinquefasciatus larvae. The effect of the lethal concentrations on C. quinquefasciatus larval mortality. The exposure time needed to achieve 30%, 50% or 90% mortality of the 3rd instar larvae of C. quinquefasciatus was determined.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 April 2015 Received in revised form 29 September 2015 Accepted 14 March 2016 Available online 16 March 2016
Efficacies of the Ammi visnaga seeds extract and a majority of substances on larval Culex quinquefasciatus mortality in various development stages including pupae were studied. The effect of exposure time on larval mortality was also studied. The effect of sublethal concentrations or short exposure times on further larval development and subsequent fecundity in adults were studied as well. Lethal doses of the extract were estimated for the 2nd, 3rd and 4th instar of C. quinquefasciatus (LC50 for 18, 23 and 180 mg L1, respectively). The majority of furanochromenes, khellin and visnagin, were identified by analysing the extract. Khellin was significantly more effective compared to visnagin, whose LC50 was estimated at 8, 10 and 41 mg L1 for the 2nd, 3rd and 4th instar larvae. Khellin showed very fast efficacy on mortality for the 3rd instar larvae in a concentration of 100 mg L1. Fifty percent mortality was determined 30 min after application, a time which was considerably shorter compared to the extract (113 min) or visnagin (169 min). The effect of the application of lethal concentrations on C. quinquefasciatus larval mortality was studied. The least number of adults were hatched after application of the extract and khellin (41.8% and 37.9%, respectively), less than after visnagin application (46.7%) or in the control (94.2%). LC50 application caused lower fecundity in the hatched adults, lower hatchability of the eggs, and also very low natality, more than 77% lower for khellin compared to the control.
Keywords: Botanical larvicide Furanochromenes Visnagin Khellin Sublethal effect Mosquito
* Corresponding author. E-mail address: [email protected] (R. Pavela). http://dx.doi.org/10.1016/j.exppara.2016.03.016 0014-4894/Crown Copyright © 2016 Published by Elsevier Inc. All rights reserved.
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A short exposure, corresponding to our estimated LT30, caused no significant acute toxicity in the larvae (until 24 h) for the extract or visnagin (4.3% and 11.5%, respectively); however, 18 min of action from khellin caused a 54.3% mortality rate of the larvae within 24 h. Crown Copyright © 2016 Published by Elsevier Inc. All rights reserved.
1. Introduction Mosquitoes are cosmopolitan and common as well as transmit a variety of diseases, while also being a nuisance. The Culex quinquefasciatus Say (Diptera: Culicidae) causes angioedema, a cutaneous allergy in humans which is also responsible for the transmission of some zoonotic pathogens (Brown, 1986; WHO, 1992). For example, C. quinquefasciatus is the dominant vector in the transmission of a number of arboviruses, or lymphatic filariasis, a disease caused by threadlike parasitic worms. Filariasis, which has infected some 120 million people worldwide, can lead to genital damage and elephantiasis (WHO, 1992; WHO, 2010). The global mosquito control strategy aims at protecting individuals and communities using long-lasting impregnated nets, indoor-residual spraying in addition to prompt and effective clinical treatment (Fillinger and Lindsay, 2011). Since long-lasting impregnated nets and indoor-residual spraying are directed against the adult vector population that enters houses, further suppression of transmission could be achieved by targeting the aquatic stages by reducing vector larval habitats, thus attacking both outdoor and indoor biting vectors. This may be particularly important in areas targeted for elimination where vector foci or 'hot spots' persist (Bejon et al., 2010; Cohen et al., 2010). The historical literature and more recent reviews of this approach show that anti-larval mosquito control measures are powerful tools against vectors. Significantly, larval source management contributed to all successful eradication efforts and successful vector-control programmes worldwide. Mosquito larvae are relatively immobile and often readily accessible. By targeting the larval stages, mosquitoes are killed ‘wholesale’ before they disperse to human habitations. Mosquito larvae, unlike adults, cannot change their habitat to avoid control activities (Fillinger and Lindsay, 2011). However, the use of insecticides faces several serious problems today. In addition to the negative effects of synthetic insecticides on the environment and non-target organisms, including man (Hodgson and Levi, 1996; Paoletti and Pimentel, 2000; Roberts and Karr, 2012), the development of resistant mosquito populations in particular is one of the most serious problems (WHO, 2012a). These problems have become the main impetus for an expeditious search for new alternatives, which would be acceptable for both the environment and health, for protection against insects. Among the existing alternative strategies aimed at decreasing vector populations, the use of pesticides based on plant extracts is currently one of the most promising. The use of botanical insecticides is a plant and vector protection alternative, generally considered safe for the environment and health (Copping and Menn, 2000; Pavela, 2014; Senthil-Nathan, 2015). Therefore, significant efforts are currently being devoted to the search for new, highly efficient plant extracts which would be suitable for the development of botanical insecticides (Miresmailli and Isman, 2014). Moreover, botanical insecticides usually contain a mixture of several active substances which exert different mechanisms of action as a rule, and thus may be able to effectively prevent the
emergence of resistant insect populations (Rattan, 2010; RegnaultRoger et al., 2012). Considerable efforts have been devoted in the past few decades to research on the biological activity of plant extracts against various insects (Pavela, 2011b; Attia et al., 2013; Senthil-Nathan, 2013; Pavela, 2013) including mosquitoes (Pavela, 2009a,b; Dias and Moraes, 2014). This research helps to find plant species that can provide active substances with a novel mechanism of effect, and are also associated with minimal health-related and environmental risks. Among these extracts, several can also be used for the development and production of botanical larvicides against important vectors (Sukumar et al., 1991; Howard et al., 2011). By comparing lethal doses of extracts obtained from plants of the Eurasian Region, several species were suitable selections for the development of botanical insecticides in one of our previous studies (Pavela, 2008, 2009a,b). The selected plants also included Ammi visnaga L. (Apiaceae), whose methanol extracts showed excellent insecticidal efficacy against C. quinquefasciatus larvae in the tests (Pavela, 2008). A. visnaga is one of the most important medicinal plants. It was used in ancient Egypt as a herbal remedy for renal colic. A. visnaga fruit preparations, such as tea prepared from crushed or powdered seeds, have traditionally been used in the Middle East to ease urinary tract pain associated with kidney stones and to promote stone passage (Gunaydin and Beyazit, 2004). Khellin has been used for the treatment of urologic, dermatologic, and respiratory symptoms. It is used in the management of bronchial asthma and angina pectoris. The plant also possesses antimicrobial activity and inhibits certain mutagens (Vedaldi et al., 1988). Taking into account that the extracts, including visnagin and khellin, have been used in popular medicine, botanical insecticides based on these substances can be considered safe for ones health. Therefore, the purpose of this study was to find more details on the biological efficacy of extracts from A. visnaga seeds and appropriate majority of substances against C. quinquefasciatus larvae. In particular: i. Efficacy of the extract and majority of substances on larval mortality at various development stages including pupae; ii. The effect of exposure time on larval mortality; and iii. The effect of sublethal concentrations or short exposure times on further larval development and subsequent fecundity in adults. The results of our work are important for estimating a possible mechanism of action and application method for A. visnaga extract as a potential larvicide. 2. Materials and methods 2.1. 1. Plant extract and analysis The seeds of A. visnaga were harvested in the autumn of 2012 from the CRI - Praha field and dried in the institute's laboratory at room temperature. Subsequently, the seeds were ground and extracted using pure methanol (MeOH) at the ratio of 1:10 (seeds: MeOH). The extraction was done for 48 h while stirring the solution from time to time; the extract was then filtered and thickened in vacuo at an operating temperature of 40 C. The obtained extract was stored in the dark, at 3 C, until the experiments. An extract
R. Pavela et al. / Experimental Parasitology 165 (2016) 51e57
sample as well as parts of the harvested plants and seeds have been stored at the RCI laboratory, Voucher No. 12009. The extract from A. visnaga was analysed by HPLC (Hewlett Packard Ti-1050) with a DAD detector (Agilent G1315B). Separation was performed on the reversed phase column (Phenomenex Luna C18, 150 2 mm, 3um) in gradient water-acetonitrile-methanolphosphoric acid at 35 C. Mobile phase A: 5% acetonitrile þ0.1% o-phosphoric acid, mobile phase B: 80% acetonitrile þ0.1% o-phosphoric acid and mobile phase C: 95% methanol. Gradient: 0. min (70% A, 20% B, 10% C), 25. min (70% A, 20% B, 10% C), 30. min (10% A, 80% B, 10% C), 35. min (90% B, 10% C), 40. min (90% B, 10% C). Flow was 0.25 ml/min. Khellin and visnagin were detected at 220 nm. Quantities of substances were calculated according to the calibration curves corresponding standards (Sigma Aldrich, Czech Republic, purity >99%). 2.2. Mosquitoes The test organism, C. quinquefasciatus, was reared in the laboratory, Secondary Plant Metabolites in Crop Protection, Crop Research Institute (Czech Republic). They were maintained at 25 ± 2 C and 75e85% relative humidity under 14:10 light and dark cycles. Pupae were transferred from the trays to a plastic cup (10 5 3 cm) containing tap water and were maintained in an insectary (45 45 40 cm) where adults emerged. Adults were maintained in plastic cages and were continuously provided with a 10% sucrose solution in a jar with a cotton wick. On day 5, the adults were given a blood meal from a chick placed in resting cages overnight for blood feeding by females. A glass petri dish with 50 ml of tap water lined with filter paper was kept inside the cage for oviposition (Pavela, 2011a). 2.3. Experimental 2.3.1. Larvicidal activity Mosquito larvicidal assays were carried out according to WHO (1996) standard procedures, with slight modifications (Pavela, 2008). The extract or furanochromenes were diluted in dimethyl sulphoxide (DMSO) to prepare a serial dilution of the test dosage. The furanochromenes visnagin and khellin, purity >98%, were obtained from Sigma Aldrich (Czech Republic) and used in the tests. Early 2nd, 3rd or 4th instar larvae of C. quinquefasciatus were selected and transferred in 224 ml of distilled water. For experimental treatment, 1 ml of serial dilutions was added to 224 ml of distilled water in a 500-ml glass bowl and shaken lightly to ensure a homogenous test solution. The selected larvae were transferred in distilled water into a bowl of prepared test solution with a final surface area of 125 cm2 (25 larvae/beaker). Four replicates were run simultaneously with at least ten dosages (from 2 to 200 mg L1). The assays were placed in a growth chamber (L16:D9, 25 ± 1 C). Mortality was determined after 24 h of exposure, during a time which no food was offered to the larvae. 2.3.2. Time needed for mortality The extract, visnagin or khellin, was applied in a concentration of 100 mg L1 to the 3rd instar larvae of C. quinquefasciatus in order to determine the activity rate of the extract and appropriate furanochromenes on larval mortality. The fundamental methodology of establishing the experiments and experimental conditions was the same as described in the previous chapter, 2.3.1. Larvicidal activity, with the only difference being that larval mortality was recorded in a time series as follows:
53
Mortality was recorded every 5 min during the first 30 min, and then at intervals of 10e15 min from 30 min to 200 min of the experiment. Any larvae that showed no signs of movement after mechanical stimulation were considered dead. In order to estimate the activity rate, 7 different time intervals were used in which mortality was determined in the range from 10% to 95%. The experiment was replicated 3 times. The assays were placed in a growth chamber (L16:D9, 25 ± 1 C). 2.3.3. Effect of lethal concentrations on larval development and fertility of surviving adults At the beginning of the 3rd instar, the larvae were put into a plastic container (20 20 20 cm) with 3 l of drinking water. Upon acclimatization (after approximately 1 h), a dose of essential oil was mixed into the water, corresponding to the calculated concentration LD50 (for the extract, visnagin and khellin, 23, 26, and 10 mg L1, respectively, Table 1). The extract or furanochromenes were emulsified using DMSO; water with an adequate DMSO content was used for the control larvae. There were 100 larvae in each container at the beginning; the assay was repeated three times. After 24 h of exposure, the larvae were transferred into clean water, where they were left until the incubation of adults. Larval mortality was determined after 24 and 48 h of exposure; total mortality, percentage of incubated adults, and their sex were determined. The larvae were fed with dog biscuits and yeast powder at a 3:1 ratio. Always 20 females and 20 males were randomly selected from incubated adults in order to determine the effect of LC50 on fecundity, fertility and natality. The adults of both sexes were placed together in breeding cages (25 25 30 cm). The adults were fed on a sugar solution, and blood feeding was provided after 7 days. A bowl with water (10 cm in diameter) was placed in the cage for oviposition. The number of oviposited eggs was determined every day (using a microscope) and a defined number of eggs was always left in order to determine their hatchability. The obtained data was used to calculate the following indicators: Fecundity ¼ E/F where E is the sum of oviposited eggs and F is the number of females that entered the experiments; Fertility (%) ¼ (H/ O)*100 where H is the number of hatched larvae and O is the total number of eggs; Natality (%) ¼ (O FEM)/L where O is the mean count of hatched larvae per female, FEM is the number of hatched females, and L is the number of larvae that entered the experiment (Pavela, 2007). The experiment was repeated 4 times. The assays were placed in a growth chamber (L16:D9, 25 ± 1 C). 2.3.4. Effect of short-term exposure on subsequent larval development and fertility of surviving adults The experiment described below was undertaken in order to determine the short-term activity of the extract or appropriate furanochromenes on larval development and subsequent fertility of surviving adults. The 3rd instar larvae of C. quinquefasciatus were exposed to a concentration of 100 mg L1 for a time period corresponding to the estimated LT30 (i.e., 75, 43 and 18 min for the extract, visnagin and khellin, respectively, see Table 2). The methodology of establishing the experiment was identical to that of 2.3.3. Effect of lethal concentrations on larval development and fertility of surviving adults, with the only difference being that after the exposure period, the larvae were transferred to clean water. The experiment was repeated 4 times. The assays were placed in a growth chamber (L16:D9, 25 ± 1 C). 2.3.5. Statistical analysis The mortality was corrected by Abbott's formula (Abbott, 1925) and an LC50, 99 and LT30, 50, 90 regression equation; a 95% confidence limit was calculated by using probit analysis (Finney, 1971).
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Table 1 Toxicity of extract and two furochromones to juvenile developmental stages of Culex quinquefasciatus during a 24 h of exposure. Extract
2nd instar 3rd instar 4th instar pupae a b
Visnagin
Khellin
LC50 (CI95)a mg.L1
LC99 (CI95)a mg.L1
Chib
LC50 (CI95)a mg.L1
LC99 (CI95)a mg.L1
Chib
LC50 (CI95)a mg.L1
LC99 (CI95)a mg.L1
Chib
18 (13e22) 23 (21e35) 180 (158e230) >200
42 (36e58) 58 (45e67) >200 >200
2.154 2.527 3.456
22 (20e25) 26 (24e28) 52 (48e59) >200
40 (35e49) 41 (37e46) 79 (65e82) >200
2.992 4.715 1.557
8 (5e9) 10 (7e12) 41 (39e49) >200
26 (22e32) 23 (21e28) 65 (59e72) >200
0.127 0.128 1.258
Ninety-five percent confidence intervals (CI), essential oils activity is considered significantly different when the 95% CI fail to overlap. Chi-square value, significant at P < 0.05 level.
Table 2 Time required to achieve 30%, 50% or 90% mortality of third instars of Culex quinquefasciatus.
Extract Visnagin Khellin
LT30 (CI95)a
LT50 (CI95)a
LT90 (CI95)a
Chib
75 (58e85) 43 (32e51) 18 (12e21)
113 (106e120) 169 (159e181) 30 (27e32)
184 (170e204) 276 (247e324) 51 (46e58)
2.929 1.266 4.388
Lethal time was calculated as time (in minutes) to indigent of 30%, 50% or 90% mortality of third instars of C. quinquefasciatus. Larvae were exposed to doses corresponding to the estimated LC99 (58, 41 and 23 mg L1 for extract, visnagin and khellin, respectively). a Ninety-five percent confidence intervals (CI), essential oils activity is considered significantly different when the 95% CI fail to overlap. b Chi-square value, significant at P < 0.05 level.
The percentage of mortality, emergence of adults and fecundity indicators were determined and transformed to arcsine square root values for analysis of variance.(ANOVA) Tukey's test (P < 0.05) was used to analyse significant differences against mosquitoes between the test extract and furanochromenes. 3. Results An extract of honey-like colour was obtained by extraction of A. visnaga seeds, with a yield of 4.87%. By analysing the extract, two majority substances belonging to the group of furanochromenes were determined: khellin and visnagin (Fig. 1), whose content was determined as 361.9 and 235.7 mg g1, respectively. In general, these two majority substances represented almost 60% of the extract content. The effect of the extract and the two determined furanochromenes on larval and pupal mortality is shown in Table 1. A significant difference was found in extract efficacies for individual development stages. While no significant difference between larvicidal efficacy of the extract was found between the 2nd and 3rd instars (LC50 ¼ 18 and 23 mg L1; LC90 ¼ 42 and 58 mg L1; respectively), the 4th instar larvae were significantly less sensitive (LC50 ¼ 180 mg L1; LC90 > 200 mg L1), and in the pupal stage, the extract caused no significant mortality (LC50 > 200 mg L1). Based on comparing the LC50 values of the furanochromenes themselves, khellin was found to be significantly more efficient against the 2nd and 3rd instar larvae of C. quinquefasciatus (LC50 ¼ 8 and 10 mg L1 for the 2nd and 3rd instar, respectively) compared to visnagin (LC50 ¼ 22 and 26 mg L1 for the 2nd and 3rd instar, respectively). In the last instar, the significance of this phenomenon decreased (P < 0.05), and none of the furanochromenes caused mortality in the pupal stage. The exposure time needed to achieve 30%, 50% or 90% mortality of the 3rd instar larvae of C. quinquefasciatus is shown in Table 2. The extract, visnagin and khellin were applied in uniform concentrations of 100 mg L1 to make sure that the time would not be affected by varying dosages. Khellin provided the fastest effect,
causing 50% and 90% mortality after 30 and 51 min, respectively, and thus in a significantly shorter time than the extract and visnagin. However, the extract provided a significantly faster effect (LT50 ¼ 113 min; LT90 ¼ 184) compared to visnagin (LT50 ¼ 169 min; LT90 ¼ 276 min). The effect of the application in lethal concentrations on C. quinquefasciatus larval mortality is presented in Table 3. Mortality 24 h after application reached approximately 50%, as expected, and kept rising only slightly during the course of larval development (by approximately 2e9%, depending on the variant). In general, significantly fewer adults hatched in all variants compared to the control. The least number of adults were hatched after the application of the extract and khellin (41.8% and 37.9%, respectively), thus less than after visnagin application (46.7%) or in the control (94.2%). LC50 application caused lower fecundity in the hatched adults (Table 3), lower hatchability of the eggs, and consequently very low natality, more than 77% lower for khellin compared to the control. The effect of the short action of the extract, visnagin and khellin on C. quinquefasciatus larvae is presented in Table 4. A short exposure, corresponding to our estimated LT30, caused no significant acute toxicity in the larvae (until 24 h) for the extract or visnagin (4.3% and 11.5%, respectively); however, 18 min of action from khellin caused a 54.3% mortality rate of the larvae within 24 h. The highest total larval mortality was recorded for khellin (65.3%) and the lowest for the extract (18.7%). Although the adults who hatched from surviving pupae showed lower fecundity after application of the extract and visnagin (Table 4), a significantly lower number of eggs per female (P > 0.05) was found only for visnagin. The effect of the extract, visnagin and khellin led to significantly lower total natality, lower by 67.6%, 53.3% and 34.8%, respectively, compared to the control. 4. Discussion In a previous study, we determined that the methanol extract from A. visnaga seeds had an excellent effect against C. quinquefasciatus larvae (Pavela, 2008). From this point of view, our current study confirms the results. However, the efficacy of the extract was newly found to significantly decrease in the last development instar of the larvae, and no effect was exerted on mortality in the pupal stage. This indicates that practical application of the extract should be aimed at early development stages of the mosquitoes. No studies of the efficacy of furanochromenes such as visnagin and khellin on mosquito larval mortality have been published until now and therefore we cannot safely compare our results with those of other authors. However, the acaricidal efficacy of an extract from A. visnaga has been known (Pavela, 2015a). The adult mortality of Tetranychus urticae was studied both for the extract from A. visnaga seeds and for visnagin and khellin. Although both of these furanochromenes were found to have a significant acaricidal effect, only visnagin showed an effect on acute toxicity (LD50 for
R. Pavela et al. / Experimental Parasitology 165 (2016) 51e57
55
DAD1 B, Sig=246,4 Ref=450,40 (ZK002934.D) mAU
175 OCH 3 O
O
150
CH3
visnagin
khellin
125
O
O
OCH 3
CH3
O
100 OCH 3
O
75
50
25
0
-25
0
5
10
15
20
25
30
35
min
Fig. 1. HPLC chromatogram of the methanolic extract of the Ammi visnaga seeds.
24 h ¼ 25 mg cm2). However, both of these substances showed a long persistence period and significant efficacy in terms of chronic toxicity. Moreover, they exhibited a very strong ovicidal efficacy (LD50 ¼ 0.5 and 1.8 mg cm2, for visnagin and khellin, respectively). The determined furanochromenes e visnagin and khellin e represented almost 60% of the extract content, and in the tests it was also demonstrated that they were responsible for the larvicidal efficacy of the extract. As for the biological efficacy of both furanochromenes, khellin was more effective than visnagin; although this significant difference declined in the last development stage, both furanochromenes also retained good efficacy against the 4th instar larvae. The furanochromenes had no effect on pupal mortality. On the contrary, as determined for the acaricidal efficacy (Pavela, 2015a), visnagin provided a much higher efficacy against all stages of T. urticae. In addition, based on our observations, the extract from A. visnaga including both of the tested substances is
not efficient against some insects (such as aphids), and it does not cause any acute or chronic toxicity (not yet published results). This would indicate that botanical insecticides based on the extract from A. visnaga are selective, which is important in terms of the environmental safety of these potential products. Comparing the lethal doses determined by us with the results of other authors, we can note that both the extract and the two active substances showed excellent biological efficacy. For the 3rd instar of C. quinquefasciatus larvae, we determined LC50 values of 23, 26 and 10 ppm (for the extract, Visnagin and Khellin, respectively). Comparing our LC50 values with the lethal concentrations of essential oils (EOs), for example, which are generally considered to have great potential for the development of botanical insecticides including larvicides (Dias and Moraes, 2014), we can note that the extract from A. visnaga shows an efficacy in terms of acute toxicity similar to the most efficient EOs. As was found (Pavela, 2015b) based on a recent evaluation of the larvicidal efficacy of EOs, only
Table 3 Effect of short time exposition of extract and furochromones on emergence and fecundity of Culex quinquefasciatus adults that survived after treatment in the larvae stage. Larval mortality (%)*
Extract Visnagin Khellin Control F, P**
Emergence of adults (%)*
Fecundity indicators*
At 24 h
At 48 h
Total
Female
Male
Total
Fecundity (No. Eggs/ female)
Fertility (% egg hatchability)
Natality (Number of larvae/ larvae)
Inhibition of natality (%)
4.3 ± 0.3b 11.5 ± 4.7c 54.3 ± 5.1d 0.0 ± 0.0a 378.27, 0.001
4.3 ± 0.3b 11.8 ± 5.1c 58.0 ± 4.4d 0.0 ± 0.0a 156.11, 0.001
18.7 ± 4.9b 27.7 ± 5.6c 65.3 ± 6.0d 2.1 ± 0.7a 201.17, 0.001
36.1 ± 2.3b 35.7 ± 5.0b 17.1 ± 1.8a 48.1 ± 0.9c 229.79, 0.001
41.0 ± 2.8c 33.8 ± 2.0b 14.5 ± 4.3a 47.5 ± 0.5d 213.29, 0.001
77.1 ± 5.1c 69.5 ± 6.0b 32.0 ± 6.1a 96.3 ± 0.4d 516.64, 0.001
145.4 ± 15.1ab 135.8 ± 21.2a 182.4 ± 11.3b 179.3 ± 21.6b 3.96, 0.035
92.6 ± 5.9 82.8 ± 20.1 87.0 ± 17.2 98.5 ± 1.3 0.12, 0.896
55.4 ± 4.5b 39.7 ± 5.6ab 27.6 ± 2.9a 85.1 ± 1.6c 39.12, 0.001
34.8 ± 4.7a 53.3 ± 5.8ab 67.6 ± 3.1b 42.05, 0.003
The 3rd instar larvae of C. quinquefasciatus were exposed to concentrations corresponding to the estimated LC99 (58, 41 and 23 mg L1 for extract, visnagin and khellin, respectively) for the time (75, 43 and 17 min for extract, visnagin and khellin, respectively) required to achieve 30% mortality of the larvae. * Mean % (±S.E.) within a column follower by the same letter do not differ significantly according to the least significant difference (LSD) test at P < 0.05 (% ¼ arc sine transformed data). ** ANOVA parameters - F-value, P-significantly level.
56
R. Pavela et al. / Experimental Parasitology 165 (2016) 51e57
Table 4 Emergence and fecundity of Culex quinquefasciatus adults that survived sublethal concentration of extract and furochromones after treatment in the larvae stage. Mortality (%)*
Extract Visnagin Khellin Control F, P**
Emergence of adults (%)*
Fecundity indicators
Total
Fecundity (no. Eggs/female)
Fertility (% egg hatchability)
Natality (number of larvae/larvae)
41.8 ± 3.1ab 46.7 ± 4.2b 37.9 ± 2.2a 94.2 ± 5.6c 178.44, 0.001
133.1 ± 25.2a 143.2 ± 21.5ab 143.6 ± 12.9a 169.9 ± 11.3b 3.68, 0.026
84.1 ± 3.2a 84.9 ± 3.6a 92.8 ± 9.2ab 98.3 ± 5.3b 4.77, 0.007
23.9 28.4 17.7 79.7 69.3
At 24 h
At 48 h
Total
Female
Male
52.6 ± 7.2b 51.1 ± 9.9b 52.6 ± 7.1b 0.0 ± 0.0a 155.59, 0.001
54.9 ± 5.3bc 51.1 ± 9.9b 58.6 ± 3.9c 0.0 ± 0.0a 346.96, 0.001
57.8 ± 4.9bc 53.1 ± 8.3b 61.63 ± 6.1c 4.4 ± 2.3a 185.66, 0.001
18.5 ± 2.5ab 23.2 ± 6.2b 13.3 ± 2.3a 51.3 ± 3.9c 44.3, 0.001
23.4 ± 23.4 ± 24.7 ± 42.8 ± 10.96, 0.001
3.8a 3.5a 2.9a 5.2c
*
± 4.2ab ± 1.7b ± 1.7a ± 5.3c 0.001
Inhibition of natality (%) 70.1 ± 5.3ab 64.4 ± 1.9a 77.7 ± 2.1b 5.37, 0.008
The 3rd instar larvae of C. quinquefasciatus were 24 h exposed to concentrations corresponding to the estimated LD50 (23, 26 and 10 mg L1 for extract, visnagin and khellin, respectively) for 24 the time (75, 43 and 17 min for extract, visnagin and khellin, respectively). * Mean % (±S.E.) within a column follower by the same letter do not differ significantly according to the least significant difference (LSD) test at P < 0.05 (% ¼ arc sine transformed data).**ANOVA parameters - F-value, P-significantly level.
122 plant species fulfilled two essential conditions: (i) LC50 100 ppm; and (ii) their chemical composition had to be known. Considering the above-estimated LC50 value as the main criterion of efficacy, 77 EOs showed LC50 < 50 ppm. Some of these efficient EOs were obtained from aromatic plants also grown commercially on relatively large areas, with a good technology of cultivation (e.g., Pimpinella anisum, Coriandrum sativum, Foeniculum vulgare, Mentha longifolia, Ocimum basilicum, Thymus spp., Eucalyptus spp., Piper spp., etc.). Only seven plants (Blumea densiflora, Auxemma glazioviana, Callitris glaucophylla, Cinnamomum microphyllum, Cinnamomum mollissimum, Cinnamomum rhyncophyllum, Zanthoxylum oxyphyllum) can be considered significantly most efficient, given that LC50 < 10 ppm has been estimated for their EOs. These EOs contained less common substances, predominantly from the group of sesquiterpenes, aromatic acids and ketones. When larvicides are applied onto a not quite stationary water surface, the applied dose may be gradually diluted by water flowing in or out, and thus their efficacy may be reduced. For this reason, the exposure time of the active substance, i.e., the time necessary to ensure the required biological efficacy, should be known. We therefore tested the activity rate for the concentration 100 mg L1, which is considered relatively acceptable for practical application (WHO, 2012b; Dias and Moraes, 2014; Bezerra-Silva et al., 2015). The extract from A. visnaga provided a relatively rapid effect, achieving 50% or 90% larval mortality within approximately 2 or 3 h after application, respectively. As for the tested furanochromenes, the most rapid effect was provided by khellin alone, which led to 90% mortality after less than 1 h from application. However, even a short exposure time or the application of lethal concentrations of the extract or its active substances showed a significant effect on overall mosquito natality in our tests. As found previously, some plant substances can lead to a significant reduction in fecundity and thus also overall natality in some vectors, even in lethal or sublethal doses or concentrations; for example, this is also the case for essential oils from Thymus vulgaris (Pavela, 2007). Our results indicate that the mechanism of effect of the A. visnaga extract is selective in terms of development stages of the mosquito, given that acute toxicity was manifested only in larval stages, while the application of the extract or furanochromenes had no effect on the pupae (not even long-term exposure to relatively high concentrations of 200 mg L1, as observed in our orientation experiments not yet published). This extract may also provide interspecies selectivity as, for example, no effect on mortality or other development characteristics in the larvae of Spodoptera littoralis (observations not yet published) has been seen in our tests. These observations of ours may indicate selectivity of the extract with respect to non-target organisms. However, further experiments will be needed to confirm this hypothesis. In conclusion, we can recommend the extract from A. visnaga
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