1 Introduction

Mosquito borne disease are the major human health problem in Sub-Saharan Africa (Nchinda, 1998; Patz et al., 2005; Smith et al., 2005; Ooi et al., 2006). The main diseases spread by mosquitoes are malaria, filariasis, chikungunya, yellow fever and hemorrhage fever (McGraw and O'Neill, 2013; Derua et al., 2015). In Tanzania among sibling species of Anopheles gambiae s.l.; An. gambiae s.s., and An. arabiensis are predominantly abundant (White and Magayuka, 1972; Mnzava and Kilama, 1986; Kweka et al., 2008c). They are potential vectors of malaria and filariasis (Derua et al., 2015). Both An. gambiae s.s., and An. arabiensis have shown to be resistant to the insecticides used currently in the public health sector (Nwane et al., 2013; Kawada et al., 2014). The resistance status has been monitored in all insecticides used for indoor residual spray (IRS) and long lasting insecticidal nets (LLINs) (Thawer et al., 2015). The existing personal protection tools needs to be improved and complemented to increase the efficiency against disease vectors. In increasing the personal protection alternative ways should be targeted for trials in different conditions to maximize their bio efficacy. In recent past, repellents have shown to add value in the reduction of human vector contact in both indoors and outdoors (Kweka et al., 2008a; Kweka et al., 2008b; Kweka et al., 2012; Karunamoorthi and Hailu, 2014). Synthetic repellents such as N, N-Diethyl-3-methylbenzamide (DEET) have shown the highest prolonged protection efficiency against disease vector (Kweka et al., 2008a; Kweka et al., 2008b; Kweka et al., 2012; Kitau et al., 2014). Menthol propylene glycol carbonate evaluated in USA and in Tanzania against flies and mosquitoes have shown high protective efficiency respectively (Kweka et al., 2012; Katunzi et al., 2015). Botanical repellents have been shown to be efficient as well after been used for long in traditional practices and in some field trials (Kweka et al., 2008a; Kweka et al., 2008b). Citronella plant extract have been regarded as the sold standard plant repellant for having long protective efficacy similarly to DEET (Kweka et al., 2008b). More compounds efficacy has to be evaluated against disease vectors for being incorporated on personal protection to increase and attain protection by IRS and LLINs.

It was the objective of this study to evaluate the protective efficiency of four products namely DEET, Maskitaa (mosquito repellent oil), Citronella and Menthol propylene glycol carbonate (MR08) against Afro-tropical malaria vectors, Anopheles gambiae sensu stricto.

2 Material and Methods

2.1 Mosquito rearing

An. gambiae s.s. used in this experiment originated from a colony of An. gambiae s.s established from Kisumu Kenya in 1992 and were reared at the tropical pesticides Research Institute (TPRI). Laboratory rearing of larvae was as described in other protocols (Tchuinkam et al., 2011; Balestrino et al., 2012). In the insectary larvae were fed with tetramin fish food at rate of 0.003 gm/larvae. The photo phase in the insectary was 12 Light: 12 Darkness (12 L: 12 D) with a temperature of 27±2ºC and Relative humidity of 78±2%. The insectary colony used is full susceptible to all insecticides approved by WHOPES for diseases vector control.

2.2 Repellents formulation and trial set up

Repellents were formulated on volume based ratio (v/v). Mixing up proportions varied from 10% (1 mls Repellent and 9 mls glycerine), 20% (2 mls Repellent and 8 mls glycerine) and 30% (3 mls Repellent and 7 mls glycerine of repellent against glycerine oil). For each repellent, all three dosages were prepared and evaluated against An. gambiae s.s. in a 30×30×30 cm cage. The arm was exposed for one hour. Each repellent experiment had six replicates.

2.3 Experimental room conditions

Repellent experiments were conducted in a room with temperature conditions 28±2ºC and humidity of 75±2%. Experimental room photo phase was 12 L: 12 D. Experimental mosquitoes were introduced in the experimental room an hour before trial.

2.4 Selection of experimental volunteers

Consent for this experiment was given to volunteers and signed by non-study participant. KOI and EJK volunteered for the repellents evaluation in all experiments.

2.5 Data analysis

Data were recorded in excel spread sheet and Transferred to SPSS 17.0 version for windows. The mean time for mosquitoes landing on the treated and untreated forearm skin was calculated using descriptive statistics for six replicates of each treatment and for control. The comparison of treatment and control arms was done using paired samples t-Test. One way Analysis of variance (ANOVA) was used to calculate the statistical difference among concentrations of repellents.

2.6 Probing and first landing time

Probing and first landing time is considered as the time taken for a mosquito to land on either treated or control hand which was measured in minutes.

2.7 Ethical issues

This study had ethical approval from tropical Pesticides research institutes. The approval was given after critical evaluation of the consent forms and validity of the study.

3 Results

3.1 Probing and first landing time

Among all repellents, Maskitaa had the extended probing time and least was Lemon grass for host seeking mosquito. DEET and Maskitaa were relatively inhibiting landing for similar time (Table 1). The variation given in probing and landing time among concentrations of the repellent and among repellents, shows Maskitaa to be a new product with competitive inhibition as good as DEET.

3.2 Protection efficiency of treatments and control

3.2.1 DEET versus control

In 10% DEET versus control, the protective efficiency was higher in DEET than in control. This was statistically significant (t=42.49, df=5, P<0.001). Protective efficiency by DEET ranged between 95% to 100% while in control was 5% to 10%. The 20% DEET lotion versus control, the protective efficiency was higher in treatment than in the control. This different in protective efficacy was statistically significant (t=42.49, df=5, P<0.001).The 30% DEET lotion versus control the protective efficacy was higher in treatment than in control. The difference was statistically significant (t=42.49, df=5, P<0.001).

3.2.2 MR08 versus control

Protection efficiency of 10% MR08 was higher than control. Protection efficiency between control and treatment was significantly different (t=18.76, df=5, P<0.001). Protection efficiency of 20% MR08 was higher than control with statistically significant difference (t=24.91, df=5, P<0.001) for the 30% MRO8, the treatment had higher protection efficiency than control. The difference was statistically different (t=58.20, df=5, P<0.001).

3.2.3 LG versus control

The 10% LG solution had higher protective efficacy than the control, which was statistically (t=34.48, df=5, P<0.001) likewise for 20% LG (t=36.66, df=5, P<0.001) and 30% LG (t=71.23, df=5, P<0.001).

3.2.4 Maskitaa versus control

The 10% Maskitaa had higher feeding protection than control which was statistically different (t=97.0, df=5, P<0.001) and 30% Maskitaa (t=97.0, df=5, P<0.001).

3.3 Comparison of repellents at different concentrations

In comparing the protection efficiency of the repellents dosages, the protective efficiency was higher in Maskitta, DEET, MRO8 and LG respectively for all dosage (Table 2).

4 Discussion

This study intended to compare the protective efficacy of four repellents against control and among them, DEET was the golden standard. The time taken for the first landing on treated and control was significantly different with treatment having extended time for the feeding attempt. This extends the protective efficacy and delays mosquito to have blood meal which subsequently reduces number of eggs laid and life span of female mosquito. In other study conducted by Wilson and others, it was revealed that, plant based extracts have the ability to extends the probing time which increases protection against infections bites (Leonidas et al., 2015). The cues emitted by repellent have found to have nerve transmitter blocking effect in such a way that mosquito cannot sense the human odour and subsequently delay feeding and affect the whole life processes (Kain et al., 2013).

DEET which is regarded as the golden standard repellent had similar effect in protection with Maskitaa, a newly formulated repellent. This shows that, if such products are screened well and in proper conditions, we can have new and effective products to protect against mosquitoes. Maskitaa reached protection efficiency of 100% similar to DEET, while LG and MRO8 were above 80% in all concentration. The use of repellents to complement the existing personal protection tool can be a great alternative for resistant mosquito population. There are reports claiming of the spreading of different mechanisms of resistance in wild population of An. gambiae s.l. (Matowo et al., 2014). There is no any reported resistance case against repellents. These plant based and synthetic repellents can have a great impact both outdoor and indoors in terms of person protection. The field trials conducted in northern Tanzania using MRO8 and DEET showed similar protective efficacy despite the reported resistance against pyrethroids in this area (Kweka et al., 2012; Matowo et al., 2014). This shows a great added value effect of repellents in personal protection in areas with high resistant strains of malaria vectors.

Despite several efforts ongoing for insecticide resistance management (Kudom et al., 2015), efforts should be directed to alternative sources such as repellents. The combination of repellents in IRS and LLINs can have positive effect in reduction of disease transmission.

The evaluated products can be very useful to target the species responsible (Aedes aegypti, Aedes albopictus and Aedes africanus) for its transmission due to recent outbreak of dengue and Zika Virus in Africa. The repellent application has to be done during the day as vectors are diurnal and bites outdoors (Baraka and Kweka, 2016). Repellents are most efficient way to control human vector contact in dengue and Zika cases reduction due to feeding behaviour of these vectors been anthropophilic.

5 Conclusion

The findings of this study have shown that, the evaluated plant based repellents have significant role in reducing human-vector contact and disease transmission and mostly for control of ZIKV and dengue transmission in Africa. More research efforts should be addressed in the search of alternative compounds to be used as repellents to add value and supplement the existing tools.

Author’s contribution

EJK designed the experiments and wrote the first draft of the manuscript. KOI, AM, EL and LL conducted experiments and data analysis. All edited and reviewed the MS and agreed upon submission.

Acknowledgement

Authors wish to thank Adrian Massawe and Ibrahim Sungi for experiment set up and data recording. TPRI for promise of infrastructure to conduct the study. There was no financial assistance for this study.

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