1 Introduction

Culex mosquitoes are vector of numbers of parasites and virus diseases that afflicted human and animals. Mosquitoes from this genus Culex are major vector of avian malaria, filariasis and arbovirus [1]. They are widely distributed across the world with exception of temperate zone of the world. Implication of Culex mosquitoes in diseases transmission call for its management, most especially in the tropical region of the world where they are more abundant. Therefore, management of this insect vector required a long-term control to reduce the menace of vector-borne diseases that afflicted human and animal [1, 2]. The use of intervention approaches is one of the most effective control measures. Adulticides, larvicides, and topical repellents among other interventions are used to interrupt vector-host interactions or contact. The life cycle of the parasitic pathogens transmitted by Culex mosquitoes is disrupted when the link between mosquito vector and human hosts are broken [2].

Management of insect-vector with synthetic insecticides has been reported as a success in the past due to their quick effect and ease of application [3, 4]. The use of synthetic pesticides or insecticides has shown to have a cognate effect on non-targeted organisms, altering their circumstances by interfering with their normal processes, and in some cases, causing the death of non-targeted organisms [5,6,7]. Over 200,000 people died every year as a result of synthetic chemicals exposure and misuse [8, 9]. Aside from the environmental issues linked with the usage of synthetic insecticides, there is also the issue of resurgence insect vector and resistance to insecticides [10,11,12]. To overcome these problems, it is necessary to sort for alternative method of control management of this insect vector. Plants derivatives have been proved by studies throughout time to efficiently control mosquitoes while posing no risk to the environment [6, 13].

Several researchers have proved the insecticidal efficiency of plant components in various studies, and these plants components are referred to as botanical insecticides. Larvicidal potency of the Thymus plant extract against Culex. pipiens pallens, Cx. quinquefasciatus, and Cx. pipiens have been reported [14,15,16]. Larvicidal activities of aromatic plants Satureja species against Cx. Pipiens had been observed [17]. Pelargonium roseum extract toxicity against Cx. pipiens was also reported [18]. Cinnamomum osmophloeum and Carum copticum extract had larvicidal activity against Cx. quinquefasciatus and Cx. pipiens, respectively [19, 20]. High toxicity activities of Cinnamomum verum, Citrus aurantifolia, Cuminum cyminum, Syzygium aromaticum, Laurus nobilis, Lippia berlandieri and Pimpinella anisum extracts against Cx. quinquefasciatus larval and pupal were also reported [21]. Citrus seeds and peels contain a variety of chemicals that have been proven to be effective against insects [22]. The dried peels of some Citrus fruits have been used in the management of pests of storage importance in a variety of methods [23]. The use of crude extract product of peels and seeds of some of Citrus fruits in the control of vectors of public health particularly vectors of parasitic diseases have recently gain recognition [24, 25]. Thus, this study is designed to evaluate the use of ethanolic extracts from four Citrus seeds species in the management of Culex mosquitoes.

2 Materials and methods

2.1 Study area

The research was conducted at the department of Biology laboratory, Federal University of Technology Akure, Ondo State. The State is located between 5° 45ʹ and 7° 52ʹ north latitude and 4° 20ʹ and 6° 05ʹ east longitude. It has a land area of around 15,500 square kilometres.

2.2 Collection of plant materials

Freshly harvested Citrus fruits (Citrus sinensis, Citrus limon, Citrus paradisi and Citrus aurantium) were purchased from commercial market and identified at the department of crop science, Federal University of Technology Akure, Ondo State.

2.3 Plant extract preparation

Before peeling, freshly obtained Citrus fruits were rinsed with distilled water. The Citrus fruit's seeds were carefully separated from the pulp. The seeds were air-dried separately for 21 days at ambient temperature (28 ± 3 °C). The dried seeds were ground into powdered materials using a commercial electric blender and stored in air-tight 250 ml transparent plastic containers. The powdered ingredients were prepared in large enough quantities (400 g) to yield up to 500 ml of extract from each Citrus species. Extraction the seed oil was carried out using a Soxhlet extractor using ethanol as the solvent. One hundred (100) grams of the powder was placed into the Soxhlet apparatus (2L) and 500 ml of ethanol was added and mixed. The mixture was heated until Citrus volatile oil was exhaustively extracted at a temperature range between 60 and 80° C for 6 h following the method described by Vogel [26] with slight modification. The extract and solvent were separated using rotary evaporator. The extracted oil was exposed at temperature (28–30° C) for the remaining ethanol to evaporate.

2.4 Collection of mosquitoes

Culex mosquito eggs/larva were collected from a slow-moving stream and allowed to hatch in the laboratory collection container. The eggs/larva were identified using the morphological keys described by Williams [27] with the use of an Olympus stereo-dissecting microscope. Before the adults emerged, the larvae were fed baker yeast and dog biscuits in the laboratory until they reached the 4th instar stage. The adult mosquitoes emerged from the pupal phases, which were transported to a rearing cage. The adult Culex mosquitoes were fed with a 10% sucrose solution.

2.5 Preparation of Citrus seeds extract concentrations

The different concentrations of the Citrus seed extract were prepared by adding 0.1 ml of the extract to 9.9 ml of distilled water to make 1% concentration of the extract as described by Simon-Oke and Akeju [24]. The following different concentrations 5%, 10%, 15%, 20%, and 25% concentrations of the extract were prepared as follows; 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml and 2.5 ml of the extract were dissolved in 9.5 ml, 9.0 ml, 8.5 ml, 8.0 ml and 7.5 ml of the distilled water respectively with three (3) replicates for each concentration, while 10 ml of distilled was used as the control (0%).

2.6 Larvicidal bioassay

The larvicidal bioassay was performed using a similar method described by WHO [28], with minor modifications. Twenty (20) 3rd/4th instar larvae from the culture were individually introduced into the various concentrations of extracted oils of the Citrus seeds, as well as a control group containing distilled water. All of the concentrations that were examined were replicated three times. An aliquot of 5 ml of the different concentrations was added to 245 ml of distilled water, while the control contains 250 ml of water. Larvae mortality was recorded after 12, 24, 36, and 48 h, the number of dead larvae was counted and recorded using the formula below:

$$\mathrm{\% \, Larval \, mortality}=\frac{\mathrm{Number \, of \, dead \, larvae}}{\mathrm{Number \, of \, larvae \, introduced}}\times 100$$
$$\mathrm{\% Corrected \, mortality}=\frac{\mathrm{\%test \, mortality}-\mathrm{\%control \, mortality}}{100-\mathrm{ \%control \, mortality}}\times 100$$

2.7 Adulticidal bioassay

The method for mosquito adulticidal test described by WHO [29] and Simon-Oke and Akeju [24] with slightly modified was adopted in this study. Twenty (20) adults were placed in a 1500 ml container with perforated top. An aliquot of 5 ml of 5%, 10%, 15%, 20%, and 25% of the Cirtus seeds extracts was soaked in cotton wools and placed in the containers containing adult Culex mosquitoes. This experiment was replicated three times. The number of dead mosquitoes was recorded after 30, 60, 90 and 120, 180 min. Percentage mortality was calculated using the formula below:

$$\mathrm{\% \, Adult \, mortality}=\frac{\mathrm{Number \, of \, dead \, adults}}{\mathrm{Number \, of \, adults \, introduced}}\times 100$$

2.8 Data analysis

The results of the adulticidal and larvicidal bioassays were subjected to one-way analysis of variance (ANOVA) and Duncan’s New Multiple Range Test (DNMRT) was used to separate the means. For bioassays where percentage mortalities were the values reported, Probit analysis was used to derive lethal concentrations (LC) required to induce 50% and 90% (LC50 and LC90) population mortality.

3 Results

3.1 Larvicidal potency of Citrus seed extract on Culex mosquitoes larval

The larvicidal effect of Citrus seed extract on larval of Culex mosquitoes is presented in Table 1. The larvicidal effect of the extract was time and concentration-dependent. The mortality of Culex mosquitoes larval increased as the period of exposure and concentration increases. After 12 h of application, 1.0% concentration of C. sinesis and C. aurantium seed oil evoked the highest larval mortality (20%) followed by C. paradisi (13%) and C. aurantifolia (10%). The effectiveness of 1.0% concentration of all the Citrus seed extracts, 1.5%, 2.0% and 2.5% of C. aurantifolia and 1.5% concentration of C. paradisi were not different significantly (p > 0.05) after 12 h of application. The highest larval mortality was recorded after 12 and 24 h of exposure from 3.0% concentration of C. sinesis and C. aurantium, 40% and 63.33% mortality were recorded respectively; this larvicidal effectiveness was not statistically difference (p > 0.05) from 1.5%, 2.0%, 2.5% concentration of C. sinesis, C. aurantium, C. paradisi, and 2.5%, 3.0% concentration of C. aurantifolia. Citrus sinesis 30% concentration evoked the highest larval mortality of 83.33% after 36 h of exposure, this value was not significantly difference (p > 0.05) from larval mortality evoked by other concentrations of Citrus seeds extract except for 1.0% and 1.5% concentration of C. aurantifolia seed extract. After 48 h of exposure, all the Citrus seed extract concentrations evoked 90% larval mortality and above which was significantly difference (p < 0.05) from the control.

Table 1 Larvicidal effect of some Citrus seeds extract on Culex mosquitoes larval
Full size table

3.2 Lethal concentration of Citrus seed extract needed to achieve 50% and 90% mortality of Culex mosquitoes larval

The concentration of Citrus seed oil needed to obtained 50% (LC50) and 90% (LC90) mortality response of Culex mosquitoes larval is presented in Table 2. Estimated lethal concentration (LC) to require to evoked 50% and 90% Culex larval mortality varies with respect to the different Citrus seeds extract. The seed extract of C. sinesis and C. aurantium has the lowest estimated lethal concentration of 4.84% to evoked 50% (LC50) Culex larval mortality within the population of 20 larval for 12 h. However, seed extract of C. aurantifolia has the lowest estimated lethal concentration of 0.03% to evoked 50% mortality (LC50) in population of 20 Culex larval for the exposure period of 48 h. The lowest lethal concentration of 38.58% and 0.95% concentration was estimated from the effectiveness of C. paradisi to evoked 90% mortality (LC90) in population of 20 Culex mosquitoes larval for 12 and 48 h exposure period respectively.

Table 2 Lethal concentration (LC50 and LC90) of the larvicidal potency of Citrus seed extract on Culex mosquito
Full size table

3.3 Adulticidal potency of Citrus seed extracted on against of adult Culex mosquitoes

The adulticidal activities of Citrus seed extract on Culex mosquitoes is represented in Table 3. The adulticidal potency of the Citrus seed extract was concentration and time depended. There is no significant difference (p > 0.05) in Culex mosquito mortality recorded from the use of 0.5% seed extract concentration of C. sinesis, C. aurantifolia, C. aurantium, and 1.0% seed extract of C. aurantifolia, C. aurantium, and control after 30 min of application. The highest mortality (75%) of adult Culex mosquitoes after 30 min of application was observed from 2.5% concentration of C. sinesis seeds extract; this value was different significantly (p < 0.0) from 2.5% concentration of others Citrus seeds extract. Culex mortality recorded from 1.0%, 1.5% and 2.0% concentration of C. sinesis, C. aurantifolia, and C. paradisi are not significantly difference (p > 0.05) after 60 min of application with the highest percentage mortality of 53.33% recorded from 2.0% concentration of C. sinesis and C. aurantifolia seed extracts. The 2.5% concentration of C. sinesis seed extract of evoked the highest Culex mosquito mortality (91.67%) after 60 min of application, this was significantly higher compare to the mortality recorded from the application of 2.5% C. aurantifolia, C. aurantium and C. paradisi. After 90 min of Citrus seed extract adulticidal assay application, the mortality (90%) observed from the application of 2.0% C. aurantifolia seed extract was higher compared to others Citrus seeds extract; however, this value was not statistically difference (p > 0.05) from mortality of adult Culex mosquito observed from C. sinesis (80%), C. aurantium (71.67%), and C. paradisi (81.67%). The adult mortality of 93.33% and 100% was observed from 2.5% concentration of C. sinesis after 90 min and 120 min respectively. There is not significant difference (p > 0.05) in percentage mortality recorded from 2.5% concentration of all the Citrus seed extracts after 90 min. C. sinesis, C. aurantifolia and C. paradisi 2.5% seed extract evoked 100% mortality after 120 min of application.

Table 3 Adulticidal effect of Citrus species seeds extract on Culex mosquitoes
Full size table

3.4 Lethal concentration of Citrus seed extract required to achieve 50% and 90% mortality of adult Culex mosquitoes

Table 4 show the estimation of Citrus seed extracted needed to achieve 50% (LC50) and 90% (LC90) population mortality of adult Culex mosquitoes with respect to time required. There is a sharp decrease in estimated concentration of Citrus seed extracts required to LC50 and LC90 as the time of exposure decreases. About 2.35%, 3.22%, 10.68%, and 11.16% concentration of C. sinesis, C. aurantium, C. paradisi, and C. aurantifolia is required respectively to achieve 50% (LC50) population mortality of adult Culex mosquitoes within 30 min of application. The estimated concentration of C. paradisi (951.67%) and C. aurantifolia (174.25%) seed extract needed in 30 min to achieve 90% (LC90) mortality of adult Culex mosquitoes is extremely high compared to concentration of C. sinesis (9.11%) and C. aurantium (8.64%) seed extract to achieve the same mortality for the same time of exposure. The lethal concentration of C. sinesis seeds extracts need to for 120 min to achieved 50% and 90% adult mosquito mortality were 0.04% and 0.54% respectively; this estimated concentration is slightly higher when compared with C. aurantifolia (0.02% and 0.40%) seed extracts concentration required to achieve the same mortality of Culex mosquito. C. paradisi seed extract lethal concentration required to evoked 50% mortality of adult Culex mosquitoes were 1.30%, 0.14%, and 0.002% for the exposure period of 60 min, 90 min, and 120 min respectively.

Table 4 Lethal concentration (LC) of the adulticidal potency of Citrus seed extract on Culex mosquito
Full size table

4 Discussion

Plant-based insecticide is the safest insecticide for the control of insect vector. This is due to their less toxic to human and environment compared to synthetic insecticides. Though, synthetic insecticides are most preferred because of their fast and quick action in eliminating vectors of parasitic diseases, but the long-term use of synthetic insecticides has been reported to be toxic to human health, contribute to insect resistance, destabilization of ecosystem and many more [30, 31]. Extract from four Citrus seed (C. sinesis, C. aurantium, C. aurantifolia, and C. paradisi) were evaluated in this study to show adulticidal and larvicidal potency in the control of Culex species mosquitoes. All the Citrus seed extracts are effective adulticides and larvicides, though the effectiveness varies based on the period of exposure, Citrus species seed, adult and larval of Culex species mosquitoes used in this study. Larval and adult Culex mosquitoes exposed to higher concentration of Citrus seed extract gave the range between 90 to 100% mortality. This finding was in agreement with the report of Sanei-Dehkordi et al. [32] and Sarma et al. [33].

C. sinesis and C. aurantium are more effective as larvicidal than adulticidal in controlling of Culex mosquitoes. Mohamed et al. [34] reported the larvicidal activities of C. sinesis and some other plants against Culex pipiens larval; the study stated out the importance of phytochemical contents from plant that induced larvicidal activities and adult mosquito repellent potency against Cx. pipiens. In a study carried out by Simon-Oke and Akeju [24], extract from peels and seeds were used to control adult Anopheles mosquitoes. The insecticidal activities of Citrus seeds extract in this study was in agreement with the result of Simon-Oke and Akeju [24]. The result of larvicidal study of C. sinesis extract reported by Sattar et al. [35] showed that the mortality responses of Cx quinquefasciatus larval to Citrus seed extract is concentration and time depended. The result of this study is similar to the findings of Sattar et al. [35]. Larvicidal potency of C. sinesis essential extract and its synergetic effect when combine with other plant extracts against Musa domestica and Anopheles stephensi has been reported by Chauhan et al. [36], it was confirmed in their study that extracted oil from C. sinesis have high potency when combine with other plant extracts for vector control. Insecticidal and repellent activity of C. aurantium essential oil against Aedes aegypti, the main vector of arbovirus has been reported by Leyva et al. [37]; the result of the study showed the effectiveness of oil extracted from C. aurantium by causing inhibition of adult mosquito emergency when the mosquito larval was exposed to LC90 of the extracted oil. However, the oil is less effective as adulticidal and repellent at a low concentration, but with higher concentration the C. aurantium oil perform excellently as adulticidal. The result of this present study was in agreement with the report of Leyva et al. [37].

The result of this study revealed that C. paradisi and C. aurantifolia are more potent in the control of adult Culex mosquitoes and less potent for the control of larval stage. However, the potency of all the Citrus seed extracts was found to be concentration dependent. Sarma et al. [33] reported that C. aurantifolia oil possess more ovicidal activity than larvicidal activity, but the larvicidal effect increase rapidly as the concentration and time of exposure increases. The findings of the study by Sarma et al. [33] collaborated outcome of the larvicidal effect of C. aurantifolia in this present study.

5 Conclusion

Larvicidal and adulticidal potency of Citrus seeds extract against larval and adult Culex mosquitoes has been established in this research. The toxicity effect of all the four Citrus seed extract are concentration and time dependent. Among the four Citrus fruits seed investigated, C. sinesis seed extract is more effective as larvicidal and adulticidal, while Citrus paradisi is more effective as adulticidal than larvicidal. These Citrus fruits seeds extract which otherwise regarded as waste could serve as promising plant-based insecticides for the control of insects of public health importance.