These Authors contrubuted equally to this work.

#### **References**

[1] Heinz FX & Stiasny K. Flaviviruses and flavivirus vaccines. Vaccine. 2012; 30(29): 4301–4306.

[2] Santhosh SR, Dash PK, Parida MM, Khan M, Tiwari M, Rao PVL. Comparative full genome analysis revealed E1: A226V shift in 2007 Indian Chikungunya virus isolates. Vir Res. 2008; 135:36–41.

infections. Also, the clinically suspected cases should be tested for both the pathogens in the endemic areas. This information is essential for early and timely diagnosis of the infecting pathogen and correlation of the clinical symptoms with mono or dual infections for appro‐ priate patient management. It has been postulated that a recent increase in Dengue and Chikungunya virus co-infection may affect the evolution of these rapidly emerging viruses. In addition, the infectivity as well as the pathogenicity of these viruses may also be affected in future. Further, continuous surveillance for both Dengue and Chikungunya viruses is essential in the endemic areas for identification and characterization of these viral pathogens. This information will also help in the implementation of proper measures to control the outbreaks

We acknowledge the financial support of University Grants Commission and Council of

1 Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi,

3 Department of Microbiology, Faculty of Medicine and Health Science, Shree Guru Gobind

[1] Heinz FX & Stiasny K. Flaviviruses and flavivirus vaccines. Vaccine. 2012; 30(29):

4 Protein Research Chair, Department of Biochemistry, College of Science, King Saud

, Irshad Hussain Naqvi2

, Shobha Broor3

,

caused by these emerging viral pathogens.

Scientific and Industrial Research, Government of India.

Farah Deeba1 #, Nazia Afreen1 #, Asimul Islam1

Singh Tricentenary University, Haryana, India

These Authors contrubuted equally to this work.

University, Riyadh, Saudi Arabia

and Shama Parveen1\*

\*Address all correspondence to: shamp25@yahoo.com, sparveen2@jmi.ac.in

2 Dr. M.A. Ansari Health Centre, Jamia Millia Islamia, New Delhi, India

**Acknowledgements**

114 Current Topics in Chikungunya

**Author details**

Anwar Ahmed4

India

#

**References**

4301–4306.


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**Section 2**

## **Entomology**

## **Vector Control in Chikungunya and Other Arboviruses**

Sengodan Karthi and Muthugounder Subramaniam Shivakumar

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63134

#### **Abstract**

Mosquito vectors are solely responsible for transmitting diseases, such as malaria, yellow fever, chikungunya, dengue, Japanese encephalitis, lymphatic filariasis and zika virus. Mosquito borne diseases are a leading killer of people and animals in developing coun‐ tries. The resurgence of diseases and the economic impact caused has brought mosquito control to the forefront. There are 3 mosquitos' genera which are vectors of these diseas‐ es, viz. Anopheles, Aedes and Culex, among these the day biting mosquito *Ae. aegypti* and *Ae. albopictus* has become important vectors to two important disease namely Dengue and Zika virus. These diseases have alone been responsible for bringing about morbidity in the large population around the world. *Cx. quinquefasciatus* is a vector of Chikungunya, which is a viral affection. It's widely spared distribution across various countries. Malaria caused by *An. stephensi* and *An. arabiensis* still affects large population in developing world. For control of emerging and reemerging mosquito borne diseases, a sound inte‐ grated approach towards comprehensive control is the need of hour which could pro‐ duce sustained effect. The reemergence of mosquito borne diseases like Zika, DHF and CHIKV coupled with the problem of insecticide resistance has both posed a danger as well as a challenge towards mosquito control. In future novel technologies especially Wolbachia based mosquito control, pesticide nanoemulsions, identification of novel bio‐ active molecules, and novel bacterial pathogens are the key to success of vector control.

**Keywords:** Mosquito vector, Zika, Wolbachia, Chikungunya, Biological control

#### **1. Introduction**

Vector-borne diseases are medically and economically important to the well being of humans and domesticated animals. In the past these diseases were considered to affect the humans living in tropical environment, but today with these diseases have a cosmopolitan distribution virtually encompassing the entire continent including temperate countries [1]. Vector borne diseases have a major impact on the economic well being of human population. A recent WHO

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

report estimates the economic loss due to vector borne diseases worldwide is to the tune of more than 2.5 billion people in over 100 countries are at risk of contracting dengue alone [30].

There are several parasites transmitted by mosquitoes and other insects. The most common transmitters being mosquitoes, which are vectors of several diseases like malaria, zika, dengue, filariasis, chikungunya, Japanese encephalitis, and yellow fever. Three mosquito genera, viz. Anopheles, Aedes and Culex are primarily the vectors which transmit these diseases. Among these diseases Chikungunya is an arthropod-borne virus transmitted mainly by *Aedes* species mosquitoes. Only the female are infective since they need blood meals for egg formation. Chikungunya virus (CHIKV) is an alphavirus from the *Togaviridae*family, which is transmitted by Aedes mosquitoes, and causes epidemic tropical and subtropical countries [28].

CHIKV is a positive-sense, single-stranded RNA virus of about 11.8 kb. There are three main genotypes, West African, Central/East African (C/EA), and Asian, the names reflecting the initial geo-graphic restriction of each type. Since CHIKV was first isolated in Tanzania in 1952 [23], the disease was mainly confined to localized outbreaks in Asia and Africa, sometimes with hundreds of thousands of cases [6]. In 2005, a C/EA strain of CHIKV, which likely originated in coastal Kenya, spread to islands of the Indian Ocean and India [19]. Since then, CHIKV spread worldwide to cause large out breaks in Southeast and East Asia, as well as many imported cases in travelers returning to non-endemic regions in Europe and North America. This global epidemic affecting millions was unprecedented in its scale and was likely driven by several factors. These include the increased volume of travellers, the widening geographic distribution of the mosquito vectors and the adaptation of the epidemic strain to *Aedes albopictus* [26]. The susceptibility of mosquitoes in non-endemic regions such as Australia [27] and North America [21], and the occurrence of autochthonous outbreaks in Italy [22] and France [10], have shown that CHIKV can no longer be considered as a problem of tropical countries. A recent work has also pointed that CHIKV may be present in severe forms, particularly neurological, which may be fatal [15]. It is also increasingly recognized that there is a significant burden of long-term morbidity due to chronic arthralgia [25]. Chikungunya is currently considered as a real threat in these European and American countries, which are colonized by *Aedes* species mosquitoes [2].

There has been little progress in finding cure for the disease. Chikungunya fever is currently symptomatically treated with antipyretics and non-steroidal anti-inflammatory drugs. There will be a continued search for new antiviral candidates with a clearly defined mechanism of viral inhibition in cell-based systems and significant activity in animal models. Considering the global threat this disease poses the immediate disease control measures solely lies in controlling mosquito population.

Dick, Kitchen and Haddow (1952) reported the first isolations of Zika virus from the blood of a sentinel rhesus monkey and from a pool of *Aedes africanus* mosquitoes in Zika forest, Uganda. Virus surveillance elsewhere has yielded more Zika virus isolations from other species of mosquitoes (Lee, 1969). Zika virus (ZIKV), a little known arbovirus, has gained prominence when it caused a large scale epidemic in the Pacific Island in 2007 [9]. It is a member of the genus *Flavivirus* of the Family Flaviviridae. The virus is a positive single stranded RNA virus with a 10,794 nucleotide genome that is closely related to the Spondweni virus [7]. It was first isolated from a febrile sentinel monkey in Uganda in 1947, but human ZIKV infection was first reported in 1964. The virus causes dengue-like syndromes such as rash, fever, arthralgia, headache and periorbital pain [24].

Zika is a tropical disease transmitted by day biting mosquito *Aedes aegypti* and like CHIKV was a largely neglected disease, until its high epidemic potential was demonstrated in pacific island of YAP in 2007 [9]. Currently this virus has made its presence in Central and South American countries in 2015 and in Feburary 2016 WHO declared Zika infections as public health emergency [32]. Like many vector-borne diseases, the absence of vaccines and specific treatment against ZIKV means prevention and control relies heavily on vector control measure which is needed in order to develop and implement sound mosquito control program.

### **2. Integrated vector control**

report estimates the economic loss due to vector borne diseases worldwide is to the tune of more than 2.5 billion people in over 100 countries are at risk of contracting dengue alone [30]. There are several parasites transmitted by mosquitoes and other insects. The most common transmitters being mosquitoes, which are vectors of several diseases like malaria, zika, dengue, filariasis, chikungunya, Japanese encephalitis, and yellow fever. Three mosquito genera, viz. Anopheles, Aedes and Culex are primarily the vectors which transmit these diseases. Among these diseases Chikungunya is an arthropod-borne virus transmitted mainly by *Aedes* species mosquitoes. Only the female are infective since they need blood meals for egg formation. Chikungunya virus (CHIKV) is an alphavirus from the *Togaviridae*family, which is transmitted

by Aedes mosquitoes, and causes epidemic tropical and subtropical countries [28].

colonized by *Aedes* species mosquitoes [2].

controlling mosquito population.

124 Current Topics in Chikungunya

CHIKV is a positive-sense, single-stranded RNA virus of about 11.8 kb. There are three main genotypes, West African, Central/East African (C/EA), and Asian, the names reflecting the initial geo-graphic restriction of each type. Since CHIKV was first isolated in Tanzania in 1952 [23], the disease was mainly confined to localized outbreaks in Asia and Africa, sometimes with hundreds of thousands of cases [6]. In 2005, a C/EA strain of CHIKV, which likely originated in coastal Kenya, spread to islands of the Indian Ocean and India [19]. Since then, CHIKV spread worldwide to cause large out breaks in Southeast and East Asia, as well as many imported cases in travelers returning to non-endemic regions in Europe and North America. This global epidemic affecting millions was unprecedented in its scale and was likely driven by several factors. These include the increased volume of travellers, the widening geographic distribution of the mosquito vectors and the adaptation of the epidemic strain to *Aedes albopictus* [26]. The susceptibility of mosquitoes in non-endemic regions such as Australia [27] and North America [21], and the occurrence of autochthonous outbreaks in Italy [22] and France [10], have shown that CHIKV can no longer be considered as a problem of tropical countries. A recent work has also pointed that CHIKV may be present in severe forms, particularly neurological, which may be fatal [15]. It is also increasingly recognized that there is a significant burden of long-term morbidity due to chronic arthralgia [25]. Chikungunya is currently considered as a real threat in these European and American countries, which are

There has been little progress in finding cure for the disease. Chikungunya fever is currently symptomatically treated with antipyretics and non-steroidal anti-inflammatory drugs. There will be a continued search for new antiviral candidates with a clearly defined mechanism of viral inhibition in cell-based systems and significant activity in animal models. Considering the global threat this disease poses the immediate disease control measures solely lies in

Dick, Kitchen and Haddow (1952) reported the first isolations of Zika virus from the blood of a sentinel rhesus monkey and from a pool of *Aedes africanus* mosquitoes in Zika forest, Uganda. Virus surveillance elsewhere has yielded more Zika virus isolations from other species of mosquitoes (Lee, 1969). Zika virus (ZIKV), a little known arbovirus, has gained prominence when it caused a large scale epidemic in the Pacific Island in 2007 [9]. It is a member of the genus *Flavivirus* of the Family Flaviviridae. The virus is a positive single stranded RNA virus with a 10,794 nucleotide genome that is closely related to the Spondweni virus [7]. It was first Vector control remains the primary defense against diseases like malaria, dengue fever, Chikungunya, zika viral infections etc. The success of vector control relies on the assumption that vector density is related to disease transmission. Vector control and disease prevention strategies are essentially divided into larval and adult mosquito control

**1.** Removal of stagnant water/ Treatment with biocide oils to prevent egg

The best method of mosquito is preventing the development of the eggs into adult mosquitoes, by reducing the source of breeding.

**2.** Insectides-Treated Bed Nets (ITNs)

The insecticides that are used for treating bed nets kill mosquitoes, as well as other insects. Only pyrethriods insecticides are approved for use on ITNs, these insecticides have been shown to pose very low health risk to humans and other mammals.

**3.** Personal production creams/sprays

These type insecticides used as repellent for some human personal production creams for control of mosquitoes against biting.

**4.** Biological control

Potential biological control agents such as fungi, *Bacillus thuringiensis*, nematodes, parasites and kill larval mosquitoes, but they are not efficient for mosquito control and are not widely used, likewise mosquito fish have largely been in effective except few studies.

Mosquito control strategies for *Aedes aegypti*, requires special attention as it has ability to breed in polluted water in addition to fresh water. Further this mosquito bites during day as well as night. *Ae. aegypti* and *Ae. albopictus* also called as tiger mosquitoes are highly dangerous in their spectral capacity of spreading dengue fever, DHF, zika and to a certain extent chikungunya diseases among people across the world. These mosquitoes have adapted themselves to be closely associated with human habitation. These mosquitoes breed in polluted as well as freshwater. They can bite at day as well as night. Their breeding sites, including overhead tanks, water in ditches, unused tyres, container harboring water, and polluted sewage water.

The control of Aedes mosquitoes can be done by removing/cleaning waste water, emptying unused water, keeping neighbor's wood are free of open source water bodies and treatment of open water bodies with chemical/biolarvicides, various personal protection steps can be undertaken like use of skin ointment/repellent, covering your bodies with cloth during day time, use of ITNs bed nets during sleeping, households, mosquito coils and other smoke repellents can reduce biting efficacy, similarly windows, household treatment etc., reduce infection

In several countries public awareness programme are conducted by different government agencies involved in mosquito control. In addition, children in schools can be educated about the ways of controlling breeding of mosquito. These community based programme can be successful in large scale eradiation of mosquito breeding sites.

Natural biological control of mosquitoes essentially targets the larval stages. Several predatory insects nymphs like of dragonflies are like Toxorhynchites each larvae and pupa. In addition like *G. affinis, G. holbrooki*, *Guppy*, and molly, found in normal/natural stress fishes, lakes are efficient in mosquito control. These are known to be very important organisms in paddy ecosystem and other water intensive crops. In addition other non-specific predators like tadpoles and other aquatic predations can help in biological control of mosquitoes.

Chemical pesticides are used by state and local government agencies to control of public health nuisance/pests dangerous to human health. Chemical pesticides are used as a last resort, to do source reduction of mosquitoes populations or when biological control flexible biological control is not feasible, may require larvicidal treatment to prevent the emergence of adult mosquitoes [20]. Use of larvicides is less controversial than use of adulticides, although use of larvicides may lead to public concern about their effects on untargeted beneficial aquatic arthropods and vertebrates.

Effective adult mosquito control with insecticides requires small droplets that drift through areas where mosquitoes are flying. The droplets that impinge on mosquitoes provide the contact activity necessary to kill them. Adulticide applications, particularly aerial applications and thermal fogging, are quite visible and contribute to public apprehension. Ground Ultra Low Volume (ULV) application may be less alarming than aerial application but is not effective over large or inaccessible areas. This technology is being developed and needs validation under different conditions with different mosquito species before it can be universally used.

Several chemical insecticides have been used in mosquito control, primary they fall into 3 broad categories viz. carbamates, organophosphate and synthetic pyrethriods, those chemical insecticides are the most powerful tool available for control of mosquito. They are highly effective for vector control and a reliable alternative for emergency action when insect pest populations exceed the economic threshold [17]. Despite the hazards of conventional insecti‐ cides, some use is unavoidable. However, careful chemical choice and application can reduce ecological damage. An array of chemicals has been developed for the killing purpose of insects. These enter the insect body either by penetrating the cuticles or dermal entry, by inhalation into the tracheal system, or by oral ingestion into the digestive system.

Chemical insecticide may be synthetic or natural products. Natural plant derived products, usually called botanical insecticides, include: alkaloids, including nicotine from tobacco, rotenone and other rotenoids from roots of legumes, pyrethroids from flowers of *Tanacetum cinerariifolium*, neem extracts from *Azadirachta indica*. The other major classes of insecticide are synthetic carbamates (carbaryl, aldicarb, methiocarb, methomyl etc), organophosphates (e.g. chlorpyrifos, dichlorvos, malathion, dimethoate and phorate) and organochlorine (aldrin, DDT, dieldrin and endosulfan etc.). Organochlorine are stable chemicals and persistent in the environment, have a low solubility in water and moderate solubility in organic solvents and accumulate in mammalian fat body. The use of this chemical is banned in many countries and they are unsuitable for use in Integrated Pest Management (IPM). Organophosphate are being less environmentally damaging and non-persistent, are suitable for IPM [11]. Most synthetic insecticides are broad spectrum in action and act on insect nervous system. Owing to its high insecticidal activity, low mammalian toxicity and high photostability pyrethroid insecticides are considered as most successful chemical classes of insecticide [4].

Today, world economy is integrated, which has led to transnational flow of goods, people, knowledge, as well as flow of pests, pathogens and vectors across geographical range where natural control is not available. Such kind of emerging infection diseases have become resurgent like zika viral infection in South America and North America, Ebola in Africa and Dengue, in India. What we thought as diseases we controlled are given becoming a threat to people across the world today.

#### **3.** *Wolbachia* **in the vector control**

freshwater. They can bite at day as well as night. Their breeding sites, including overhead tanks, water in ditches, unused tyres, container harboring water, and polluted sewage water.

The control of Aedes mosquitoes can be done by removing/cleaning waste water, emptying unused water, keeping neighbor's wood are free of open source water bodies and treatment of open water bodies with chemical/biolarvicides, various personal protection steps can be undertaken like use of skin ointment/repellent, covering your bodies with cloth during day time, use of ITNs bed nets during sleeping, households, mosquito coils and other smoke repellents can reduce biting efficacy, similarly windows, household treatment etc., reduce

In several countries public awareness programme are conducted by different government agencies involved in mosquito control. In addition, children in schools can be educated about the ways of controlling breeding of mosquito. These community based programme can be

Natural biological control of mosquitoes essentially targets the larval stages. Several predatory insects nymphs like of dragonflies are like Toxorhynchites each larvae and pupa. In addition like *G. affinis, G. holbrooki*, *Guppy*, and molly, found in normal/natural stress fishes, lakes are efficient in mosquito control. These are known to be very important organisms in paddy ecosystem and other water intensive crops. In addition other non-specific predators like

Chemical pesticides are used by state and local government agencies to control of public health nuisance/pests dangerous to human health. Chemical pesticides are used as a last resort, to do source reduction of mosquitoes populations or when biological control flexible biological control is not feasible, may require larvicidal treatment to prevent the emergence of adult mosquitoes [20]. Use of larvicides is less controversial than use of adulticides, although use of larvicides may lead to public concern about their effects on untargeted beneficial aquatic

Effective adult mosquito control with insecticides requires small droplets that drift through areas where mosquitoes are flying. The droplets that impinge on mosquitoes provide the contact activity necessary to kill them. Adulticide applications, particularly aerial applications and thermal fogging, are quite visible and contribute to public apprehension. Ground Ultra Low Volume (ULV) application may be less alarming than aerial application but is not effective over large or inaccessible areas. This technology is being developed and needs validation under different conditions with different mosquito species before it can be universally used.

Several chemical insecticides have been used in mosquito control, primary they fall into 3 broad categories viz. carbamates, organophosphate and synthetic pyrethriods, those chemical insecticides are the most powerful tool available for control of mosquito. They are highly effective for vector control and a reliable alternative for emergency action when insect pest populations exceed the economic threshold [17]. Despite the hazards of conventional insecti‐ cides, some use is unavoidable. However, careful chemical choice and application can reduce ecological damage. An array of chemicals has been developed for the killing purpose of insects. These enter the insect body either by penetrating the cuticles or dermal entry, by inhalation

into the tracheal system, or by oral ingestion into the digestive system.

tadpoles and other aquatic predations can help in biological control of mosquitoes.

successful in large scale eradiation of mosquito breeding sites.

infection

126 Current Topics in Chikungunya

arthropods and vertebrates.

A new approach to dengue control has recently been proposed that targets mosquito longevity rather than abundance, through the introduction of a life-shortening strain of the bacterium *Wolbachia pipientis* into *Ae. aegypti* populations [5]. α- proteobacteria is best endosymbiotic bacteria species present in insects and nematodes [13]. They infect diverse several arthropods, this is known to affect reproduction and produce nutrients, provide production against pathogens [12, 14]. Wolbachia is an intracellular inherited bacterium, predicted to naturally infect more than 60% of all insect species worldwide [13] that is able to invade host populations through either the induction of a number of reproductive parasitism traits [18] or by positively influencing host fitness [3].

Wolbachia is cytoplasmically inherited, so, infected females usually give rise to females. CI occurs when infected male mates with normal female. In reserve case the offspring are female multiple wolbachia stains may be present in one insect.

Wolbachia is known reproduce parasite when produces cytoplasmic incompatibility, sex ratio distorters, parthenogenesis [29]. They can also enhance reproduce fitness [8]. Mosquito control CI incompatible mates, in *Ae. aegypti* which in naturally uninfected with wolbachia. Wolbachia infected cell lines developed by embryonic microinjection [16], cathartic produces based vector control strategy have been tested in several regions of the world. The release of Wolbachia infected males is an approach that may suppress/reduce natural mosquito population.

#### **4. Future perspective**

Vector control strategy essentially needs to encompass 4 broad strategies 1. Spreading and effective way to contain diseases outbreak. 2. Adult mosquito repellant 3. Larval control in aquatic systems 4. Personal protection gear and tools.

Environmental management is a community based approach to eradicate mosquito breeding sites in their locality. To a large extent this exercise can reduce the mosquito population and is rather an effective strategy in urban and sub-urban areas. This approach has its limitations in villages due to the presence of large open water bodies for irrigation and irrigation systems, also environment such as paddy agroecosystem is a rich reservoir of water and is an ideal breeding ground for mosquitoes natural control by entomophagous predators and predatory fishes contain mosquito population to a large extent.

Adult mosquito management using mosquito repellants, in the form of mosquito coils, mats and liquid vaporizers are still in existence; however these are not a permanent solution to adult mosquitoes as they quickly develop resistance to these insecticides in few years. Use of entomopathogenic nematodes, fungus for adult control has limited success. However a new method/ approach of recombinant genetic transformation of Wolbachia in *Aedes* and *Anophe‐ les* genera has been shown to be a promising method for disrupting mosquito –viral interaction thus can be a substantially reduce the disease progression. Personal protection creams like DEET, and other herbal products are still the better choice for person safety, ITNs have been very successful in the control of *Culex* and *Anopheles* borne diseases, however this is rather ineffective against day biting behavior of *Aedes* genera. Sterile male insect technique has met with only limited success and transgenic mosquitoes carrying dominant lethal genes is another method which is still in its infancy. Research needs to address the dynamics of inheritance of these lethal genes and it should also address the sexual selection of transgenic mosquitos' males to others in order to fully analyse its potential in mosquito control. The recent reemergence of mosquito borne diseases like Zika, DHF and CHIKV coupled with the problem of insecticide resistance has both posed a danger as well as a challenge towards mosquito control. In future novel technologies especially Wolbachia based mosquito control, nano emulsions, identifica‐ tion of novel bioactive molecules, and novel bacterial pathogens are the key to success of vector control.

#### **Author details**

Sengodan Karthi and Muthugounder Subramaniam Shivakumar\*

\*Address all correspondence to: skentomol@gmail.com

Molecular Entomology Laboratory, School of Biosciences, Department of Biotechnology, Periyar University, Salem, Tamil Nadu, India

#### **References**

**4. Future perspective**

128 Current Topics in Chikungunya

control.

**Author details**

aquatic systems 4. Personal protection gear and tools.

fishes contain mosquito population to a large extent.

Sengodan Karthi and Muthugounder Subramaniam Shivakumar\*

Molecular Entomology Laboratory, School of Biosciences, Department of Biotechnology,

\*Address all correspondence to: skentomol@gmail.com

Periyar University, Salem, Tamil Nadu, India

Vector control strategy essentially needs to encompass 4 broad strategies 1. Spreading and effective way to contain diseases outbreak. 2. Adult mosquito repellant 3. Larval control in

Environmental management is a community based approach to eradicate mosquito breeding sites in their locality. To a large extent this exercise can reduce the mosquito population and is rather an effective strategy in urban and sub-urban areas. This approach has its limitations in villages due to the presence of large open water bodies for irrigation and irrigation systems, also environment such as paddy agroecosystem is a rich reservoir of water and is an ideal breeding ground for mosquitoes natural control by entomophagous predators and predatory

Adult mosquito management using mosquito repellants, in the form of mosquito coils, mats and liquid vaporizers are still in existence; however these are not a permanent solution to adult mosquitoes as they quickly develop resistance to these insecticides in few years. Use of entomopathogenic nematodes, fungus for adult control has limited success. However a new method/ approach of recombinant genetic transformation of Wolbachia in *Aedes* and *Anophe‐ les* genera has been shown to be a promising method for disrupting mosquito –viral interaction thus can be a substantially reduce the disease progression. Personal protection creams like DEET, and other herbal products are still the better choice for person safety, ITNs have been very successful in the control of *Culex* and *Anopheles* borne diseases, however this is rather ineffective against day biting behavior of *Aedes* genera. Sterile male insect technique has met with only limited success and transgenic mosquitoes carrying dominant lethal genes is another method which is still in its infancy. Research needs to address the dynamics of inheritance of these lethal genes and it should also address the sexual selection of transgenic mosquitos' males to others in order to fully analyse its potential in mosquito control. The recent reemergence of mosquito borne diseases like Zika, DHF and CHIKV coupled with the problem of insecticide resistance has both posed a danger as well as a challenge towards mosquito control. In future novel technologies especially Wolbachia based mosquito control, nano emulsions, identifica‐ tion of novel bioactive molecules, and novel bacterial pathogens are the key to success of vector


[28] Weaver SC, Osorio JE, Livengood JA, Chen R, Stinchcomb DT. 2005. Chikungunya virus and prospects for a vaccine. Expert Rev Vaccines, 11:1087–101.

[14] Hosokawa, T., Koga, R., Kikuchi, Y., Meng, X-Y., Fukatsu, T. (2010). Wolbachia as a bacteriocyte- associated nutritional mutualist. Proc. Natl.Acad. Sci. USA, 107,

[15] Lemant J, Boisson V, Winer A, Thibault L, Andre H, Tixier F, 2008. Serious acute chi‐ kungunya virus infection requiring intensive care during the Reunion Island out‐

[16] Mc Meninman, C.J., Lane, A.M., Cass, B.N., Fong, A.W.C., Sindhu, M., Wang, Y.F., O'Neill, S.L. (2009). Stable introduction of a life shorting Wolbachia: infection into the

[17] Metcalf, R.L., Luckmann, W.H., 1994. Introduction to Insect Pest Management. New

[18] O'Neill, S.L., Hoffmann, A.A., and Werren, J.H. 1997. Influential passengers: inherit‐ ed microorganisms and arthropod reproduction (Oxford University Press)

[19] Njenga, MK, Nderitu, L, Ledermann, J. P, Ndirangu, A, Logue, CH, Kelly, CHL, Sang, R, Sergon, K, Breiman, R, Powers, AM. (2008). Tracking epidemic Chikungu‐ nya virus into the Indian Ocean from East Africa J. Gen. Virol. 89 (11): 2754–2760.

[20] Rawlings JA, Hendricks KA, Burgess CR, Campman RM, Clark GG, Tabony LJ, Pat‐ terson MA. 1998. Dengue surveillance in Texas, Am J Trop Med Hyg,; 59:95-9.

[21] Reiskind MH, Pesko K, Westbrook CJ, Mores CN. 2008. Susceptibility of Florida mos‐ quitoes to infection with chikungunya virus. Am J Trop Med Hyg, 78:422–5.

[22] Rezza G, Nicoletti L, Angelini R, Romi R, Finarelli AC, Panning M, 2007. Infection with chikungunya virus in Italy: an outbreak in a temperate region. Lancet,370:1840–

[23] Robinson MC. 1955. An epidemic of virus disease in Southern Province, Tanganyika Territory, in 1952–53. I. Clinical features. Trans R Soc Trop Med Hyg, 49:28–32.

[24] Simpson DI (1964) Zika Virus Infection in Man. Trans R Soc Trop Med Hyg 58: 335–

[25] Sissoko D, Malvy D, Ezzedine K, Renault P, Moscetti F, Ledrans M, 2009. Post-epi‐ demic Chikungunya disease on Reunion Island: course of rheumatic manifestations

[26] Tsetsarkin KA, Vanlandingham DL, McGee CE, Higgs S. 2007. A single mutation in chikungunya virus affects vector specificity and epidemic potential. PLoS Pathog, 3:

[27] Van den Hurk AF, Hall-Mendelin S, Pyke AT, Smith GA, Mackenzie JS. 2010. Vector competence of Australian mosquitoes for chikungunya virus. Vector Borne Zoonotic

and associated factors over a 15-month period. PLoS Negl Trop Dis, 3:389.

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## **Utilization of Fruit Peel Wastes for the Management of Chikungunya Vector,** *Aedes aegypti*

Sarita Kumar, Monika Mishra, Aarti Sharma and Radhika Warikoo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64430

#### **Abstract**

Chikungunya, a widely spread viral disease transmitted to human beings by *Aedes aegyp‐ ti*, is on rise in India, Africa and Asian subcontinent since last decade. Although chemical insecticides are used at a large scale for the control of Chikungunya vector, their applica‐ tions have led to several undesirable effects including insecticide resistance, revival of pests species, appearance of secondary pests, environmental pollution, noxious hazards to human beings and non-target organisms forcing investigators to explore unconven‐ tional alternate strategies. As an environment-friendly approach, there is increased atten‐ tion to devise and adopt suitable methods to utilize wastes as value-added products to reduce the problem of environmental pollution. Consequently, the larvicidal and adult ir‐ ritant potential of hexane and petroleum ether peel extracts of three different *Citrus* spe‐ cies*, C. limetta*, *C. sinensis* and *C. Limon,* were assessed against *Ae. aegypti*. The results showed the larvicidal potential of all the three peels, *C. limetta* peel extracts exhibiting the least activity. Furthermore, hexane extracts were more effective than petroleum ether ex‐ tracts, *C. sinensis* peels hexane extract being most effectual (LC50, 39.51 ppm) while petro‐ leum ether peels extract of *C. limon* was the most effective larvicide with LC50 value of 51.25 ppm. All the extracts also exhibited significant elicit response and irritant potential against adults signifying their potential role in reduced mosquito bites and disease trans‐ mission. The qualitative phytochemical analysis of the extracts showed presence of cer‐ tain components suggesting their probable role in bioefficacy of extracts. Further studies are needed to isolate and identify the active ingredient to formulate strategies for mosqui‐ to control.

**Keywords:** *Aedes aegypti*, citrus, larvicidal, peel waste, repellent

#### **1. Introduction**

Mosquitoes and the related mosquito-borne diseases are a growing menace around the globe. Prevalence of mosquito-borne diseases is one of the world's most health hazardous problems.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Several mosquito species belonging to genera *Culex*, *Anopheles* and *Aedes* are vectors for the pathogens of various diseases like filariasis, Japanese encephalitis, malaria, dengue, dengue haemorrhagic fever, yellow fever and Chikungunya [1]. *Ae. aegypti*, the primary carrier for viruses, that cause dengue and Chikungunya fever, is prevalent over wide regions of the tropical and subtropical world. Currently, no effectual vaccine is offered for dengue and Chikunguya fever. Recently, Zika virus disease transmitted by *Aedes* mosquitoes, especially *Ae. aegypti* in tropical world, has been the cause of concern in the world. In today's scenario, the only approach that can be employed to reduce the incidence of *Aedes*-borne diseases is by vector control, which is recurrently reliant on the application of conventional synthetic chemical insecticides [2].

*Ae. aegypti*, thus has engrossed extensive interest at universal level because of rapid spread of these diseases. Chikungunya is primarily centred across the urban and peri-urban areas, having severe effects in the Asian subcontinent. WHO reports state the massive-fold increase in the number of reported cases over the last decade. Also, the unavailability of a vaccine against the disease further makes the scenario graver.

The first documented outbreak of Chikungunya took place in 1952 in Southern Tanzania, East Africa [3]. Subsequently, epidemics were recorded soon in several other countries : Cambodia, Thailand, Philippines, India, Vietnam, Burma and Sri Lanka. Ever since 2003, there have been outbreaks in the islands of the Pacific Ocean, including Comoros, Mauritius, Madagascar and Reunion Island [4].

The increasing population of Chikungunya vector has urged scientists to come up with effective methods of mosquito control. Several methods have been adopted for controlling mosquito population. In the past, control measures for insect pests and disease vectors utilizing synthetic organic chemical insecticides have caused development of resistance to these insecticides in the disease vector of Chikungunya and dengue fever [5]. It has been reported that insecticide resistance may develop due to changes in the biochemical machinery of mosquitoes, resulting in the quick detoxification or appropriation of the insecticide, or because of the mutation in the target site inhibiting its interaction with insecticide [6]. Progressively, the problem of insecticide resistance is augmenting leading to collapse of many vector control programs. Moreover, the recurrent use of insecticides to manage insect pests and vectors is causing deterioration of the ecosystem [7]. These insecticides are thus, now not a preferred choice for mosquito control because of the environmental degradation they usher. Thus, scientists are now on the look-out for alternative strategies to circumvent the problems posed by the use of synthetic chemical-based insecticides.

Botanicals are one such alternative; researchers are banking upon to alleviate these problems. Although using botanicals seems to be a relatively newer form of mosquito control but its history dates back to the first century AD when a Greek philosopher, Pliny the Elder, wrote ''Natural History'' and recorded all the pest control methods known at that time. Simultane‐ ously, the Chinese discovered the use of *Chrysanthemum* flowers as an effective insecticide and many others reported a few important phytochemical insecticides, including pyrethrum, derris, quassia, nicotine, hellebore, anabasine, azadirachtin, d-limonene, camphor and turpentine, widely used earlier in developed countries before the introduction of synthetic organic insecticides.

Several mosquito species belonging to genera *Culex*, *Anopheles* and *Aedes* are vectors for the pathogens of various diseases like filariasis, Japanese encephalitis, malaria, dengue, dengue haemorrhagic fever, yellow fever and Chikungunya [1]. *Ae. aegypti*, the primary carrier for viruses, that cause dengue and Chikungunya fever, is prevalent over wide regions of the tropical and subtropical world. Currently, no effectual vaccine is offered for dengue and Chikunguya fever. Recently, Zika virus disease transmitted by *Aedes* mosquitoes, especially *Ae. aegypti* in tropical world, has been the cause of concern in the world. In today's scenario, the only approach that can be employed to reduce the incidence of *Aedes*-borne diseases is by vector control, which is recurrently reliant on the application of conventional synthetic

*Ae. aegypti*, thus has engrossed extensive interest at universal level because of rapid spread of these diseases. Chikungunya is primarily centred across the urban and peri-urban areas, having severe effects in the Asian subcontinent. WHO reports state the massive-fold increase in the number of reported cases over the last decade. Also, the unavailability of a vaccine

The first documented outbreak of Chikungunya took place in 1952 in Southern Tanzania, East Africa [3]. Subsequently, epidemics were recorded soon in several other countries : Cambodia, Thailand, Philippines, India, Vietnam, Burma and Sri Lanka. Ever since 2003, there have been outbreaks in the islands of the Pacific Ocean, including Comoros, Mauritius, Madagascar and

The increasing population of Chikungunya vector has urged scientists to come up with effective methods of mosquito control. Several methods have been adopted for controlling mosquito population. In the past, control measures for insect pests and disease vectors utilizing synthetic organic chemical insecticides have caused development of resistance to these insecticides in the disease vector of Chikungunya and dengue fever [5]. It has been reported that insecticide resistance may develop due to changes in the biochemical machinery of mosquitoes, resulting in the quick detoxification or appropriation of the insecticide, or because of the mutation in the target site inhibiting its interaction with insecticide [6]. Progressively, the problem of insecticide resistance is augmenting leading to collapse of many vector control programs. Moreover, the recurrent use of insecticides to manage insect pests and vectors is causing deterioration of the ecosystem [7]. These insecticides are thus, now not a preferred choice for mosquito control because of the environmental degradation they usher. Thus, scientists are now on the look-out for alternative strategies to circumvent the problems posed

Botanicals are one such alternative; researchers are banking upon to alleviate these problems. Although using botanicals seems to be a relatively newer form of mosquito control but its history dates back to the first century AD when a Greek philosopher, Pliny the Elder, wrote ''Natural History'' and recorded all the pest control methods known at that time. Simultane‐ ously, the Chinese discovered the use of *Chrysanthemum* flowers as an effective insecticide and many others reported a few important phytochemical insecticides, including pyrethrum, derris, quassia, nicotine, hellebore, anabasine, azadirachtin, d-limonene, camphor and

chemical insecticides [2].

134 Current Topics in Chikungunya

Reunion Island [4].

against the disease further makes the scenario graver.

by the use of synthetic chemical-based insecticides.

Though with the advent of DDT in 1939, the researchers' quest for mosquito control agents was over and phytochemicals saw their downfall, yet there was a re-focus on phytochemicals because of several problems faced due to injudicious and over application of synthetic insecticides in nature. The scientists have always appreciated the easily biodegradable nature of the phytochemicals leading to no ill-effects on non-target organism. Almost two decades back, phytochemicals could make up only 1 % of world's pesticide market but now, there is a continuous rise in the use of these environment-friendly chemicals.

By definition, phytochemicals are non-nutritive plant chemicals with defensive characteristics. They assist the plants to endure the incessant selection pressure from herbivorous pests, predators and other environmental factors. They are known to be the chemical factories of nature, producing many chemicals, some of which have medicinal and pesticidal properties [8]. Researchers have identified several groups of phytochemicals which possess insecticidal activities. It has been reported that approximately 2000 plant species produce secondary metabolites of importance in pest control programs [9]. Amongst these plant species, metab‐ olites of approximately 344 species have been reported to have a range of activity against mosquitoes [10]. In 2000, Liu et al. suggested these natural plant products as possible alterna‐ tives to synthetic chemical insecticides [11]. Some of such phytochemicals include alkaloids, steroids, terpenoids, essential oils and phenolic compounds. The intensity of the insecticidal effect of a particular plant is not static and varies with plant species, mosquito species, geographical varieties, part of the plant used, extraction method adopted, etc. Bio-pesticides provide an alternative to synthetic pesticides as they are effective, easily available, safe for manufacturers and users, biodegradable, inexpensive and environment-friendly.

Recently, there is always an increased attention in bringing useful products from waste materials and fruit wastes are no exceptions. With the increase in production of processed fruit products, the amount of fruit wastes generated is increasing enormously. Citrus is one of the most important commercial fruit crops grown in all continents of the world, the fruits primarily used by juice processing industries while the peels are considered a waste material [12]. Since the juice yield of citrus is less half of the fruit weight, enormous quantity of peel wastes are formed every year. Unfortunately, the peels though perishable and seasonal, they pose a big problem of disposal to the processing industries and pollution monitoring agencies. There is a growing interest to adopt appropriate methods for utilizing them for the conversion into value-added products and improve the overall economics of processing units and reduce the problem of environmental pollution considerably.

Recycling of fruit waste is one of the most imperative means of exploiting it in a number of pioneering and ground-breaking ways leading to the yield of novel products and meeting the requirements of indispensable products required in pharmaceutical industry. The reports are available which confirmed the presence of nutrients and several secondary metabolites in the citrus peels in high magnitude. This clearly indicates the probable use of citrus peel products as drugs and harmless alternate to synthetic chemicals. Consequently, citrus plants have attracted the attention of many workers, and thus organic extracts or essential oils of the leaves

and fruit peels are under investigations for various purposes, especially against control of mosquitoes. Suitable methods have to be adopted to utilize them for the conversion into valueadded products to improve the overall economics of processing units and reduce the problem of environmental pollution considerably [13]. Many plants have been found to contain chemicals, which are helpful for the control of insects and are useful for field applications in mosquito control programs [14]. Limonene isolated from abraded fresh peels of *Citrus aurantium* has been investigated for its toxicity against larvae of *Cx. quinquefasciatus* and found effective [15]. In 2006, Lee assessed the larvicidal efficacy of *Citrus bergamia* essential oil against *Ae. aegypti* and *Cx. Pipiens* [16], while Amer and Mehlhorn investigated essential oil extracted from *C. limon* against three mosquito species : *Ae. aegypti*, *Cx. quinquefasciatus* and *Anopheles stephensi* [17].

It is well known that the approach to combat mosquito-borne diseases largely relies on interruption of the disease transmission cycle by either targeting the mosquito larvae at breeding sites through spraying of stagnant water or by killing/repelling the adult mosquitoes using insecticides. Thus, investigations were carried out to explore the efficient anti-mosquito potential of citrus peels obtained from three species of *Citrus* : *C. limetta*, *C. limon* and *C. sinensis* to control *Ae. aegypti* at larval stage and to repel at adult stage keeping in mind that such a study would not only devise alternate strategies for mosquito control but also would focus towards recycling of wastes, managing littering and waste-related environmental degradation.

#### **2. Review of literature**

Citrus plants have attracted the attention of many scientists. Consequently, researchers have carried out investigations on the organic extracts and essential oils extracted from the leaves and fruit peels of citrus plants against different species of insect pests including mosquitoes [18‒21]. There are reports available which confirm the larvicidal and repellent activities of the essential oil isolated from *C. sinensis* against *Ae. aegypti* mosquitoes [22].

The larvicidal efficacy of *C. sinensis* peels has been documented against various species of mosquitoes [21]. They reported the respective LC50 and LC90 values of 58.25 and 298.31 ppm when the larvae of *An*. *subpictus* were exposed to the chloroform extract of *C*. *sinensis* peels. On the other hand, assays with methanol extract of *C*. *sinensis* peels resulted in lower LC50 and LC90 values of 38.15 and 184.67 ppm, respectively against the larvae of *Cx*. *tritaeniorhynchus*. The effectiveness of the ethanolic extracts of the *C. sinensis* has been established by Amusan et al. against the larvae of *Ae. aegypti* [23]. Likewise, Michaelakis et al. reported the strong toxicity of essential oils extracted from peels of *C. limon* and *C. sinensis* against larvae of *Cx. pipiens* resulting in the LC50 values ranging from 30.1 to 51.5 mg/l, respectively [24]. Earlier investi‐ gations have shown that the peel oil extracts of *C. sinensis*, *C. aurantium* and *C*. *limon* possessed outstanding larvicidal potential against *Cx. quinquefasciatus* with lemon peel oil exhibiting the maximum larvicidal efficacy [18,19]. The volatile extracts of *C. sinensis* and *C. aurantifolia* (lime) peels were also observed to have cidal activity against mosquitoes [25].

It has been reported earlier that phytochemicals play a foremost role in the mosquito control programmes. Different secondary metabolites of plants, such as alkaloids, steroids, phenolics, terpenoids, saponins, essential oil, etc., are coupled with an extensive range of biological activities. Marston et al. have reported the efficacy of prenylated xanthones, tetracyclic phenols and saponins in controlling the mosquito *Ae. aegypti* [26]. Likewise, the presence of carbohy‐ drates, saponins, phytosterols, phenols, flavonoids and tannins has been observed in the plant extract exhibiting mosquito larvicidal activity [27]. Furthermore, the citrus fruit peel has been found to be a rich source of flavanones and many polymethoxylated flavones, rare in other plants [28]. Kumar et al. have showed the presence of alkaloids, flavonoids and terpenoids in the petroleum ether extracts of *C. sinensis* peels [29]. Table 1 summarizes the larvicidal activity of certain *Citrus* species against different species of mosquitoes.


**Table 1.** Larvicidal activity of certain *Citrus* species against different species of mosquitoes

#### **3. Materials and methods**

and fruit peels are under investigations for various purposes, especially against control of mosquitoes. Suitable methods have to be adopted to utilize them for the conversion into valueadded products to improve the overall economics of processing units and reduce the problem of environmental pollution considerably [13]. Many plants have been found to contain chemicals, which are helpful for the control of insects and are useful for field applications in mosquito control programs [14]. Limonene isolated from abraded fresh peels of *Citrus aurantium* has been investigated for its toxicity against larvae of *Cx. quinquefasciatus* and found effective [15]. In 2006, Lee assessed the larvicidal efficacy of *Citrus bergamia* essential oil against *Ae. aegypti* and *Cx. Pipiens* [16], while Amer and Mehlhorn investigated essential oil extracted from *C. limon* against three mosquito species : *Ae. aegypti*, *Cx. quinquefasciatus* and *Anopheles*

It is well known that the approach to combat mosquito-borne diseases largely relies on interruption of the disease transmission cycle by either targeting the mosquito larvae at breeding sites through spraying of stagnant water or by killing/repelling the adult mosquitoes using insecticides. Thus, investigations were carried out to explore the efficient anti-mosquito potential of citrus peels obtained from three species of *Citrus* : *C. limetta*, *C. limon* and *C. sinensis* to control *Ae. aegypti* at larval stage and to repel at adult stage keeping in mind that such a study would not only devise alternate strategies for mosquito control but also would focus towards recycling of wastes, managing littering and waste-related environmental

Citrus plants have attracted the attention of many scientists. Consequently, researchers have carried out investigations on the organic extracts and essential oils extracted from the leaves and fruit peels of citrus plants against different species of insect pests including mosquitoes [18‒21]. There are reports available which confirm the larvicidal and repellent activities of the

The larvicidal efficacy of *C. sinensis* peels has been documented against various species of mosquitoes [21]. They reported the respective LC50 and LC90 values of 58.25 and 298.31 ppm when the larvae of *An*. *subpictus* were exposed to the chloroform extract of *C*. *sinensis* peels. On the other hand, assays with methanol extract of *C*. *sinensis* peels resulted in lower LC50 and LC90 values of 38.15 and 184.67 ppm, respectively against the larvae of *Cx*. *tritaeniorhynchus*. The effectiveness of the ethanolic extracts of the *C. sinensis* has been established by Amusan et al. against the larvae of *Ae. aegypti* [23]. Likewise, Michaelakis et al. reported the strong toxicity of essential oils extracted from peels of *C. limon* and *C. sinensis* against larvae of *Cx. pipiens* resulting in the LC50 values ranging from 30.1 to 51.5 mg/l, respectively [24]. Earlier investi‐ gations have shown that the peel oil extracts of *C. sinensis*, *C. aurantium* and *C*. *limon* possessed outstanding larvicidal potential against *Cx. quinquefasciatus* with lemon peel oil exhibiting the maximum larvicidal efficacy [18,19]. The volatile extracts of *C. sinensis* and *C. aurantifolia* (lime)

essential oil isolated from *C. sinensis* against *Ae. aegypti* mosquitoes [22].

peels were also observed to have cidal activity against mosquitoes [25].

*stephensi* [17].

136 Current Topics in Chikungunya

degradation.

**2. Review of literature**

#### **3.1. Collection of the larvae and adults of** *Ae. aegypti*

The present investigations employ the Chikungunya fever mosquito, *Ae. aegypti* that originat‐ ed from fields of Delhi and surrounding areas. The larvae were collected from seven locations spread across Delhi and NCR (National Capital Region) (Figure 1). The locations selected were:


The larvae and adults collected from different locations were brought to the laboratory. The stages were critically observed and those of *Aedes* were segregated and bought back to the laboratory for rearing.

#### **3.2. Laboratory colonization and maintenance of** *Ae. aegypti*

The colony of *Ae. aegypti* was maintained in an insectary under controlled conditions of 28 ± 1°C, 80 ± 5% relative humidity and 14:10 light/ dark photoperiod [30]. Adult mosquitoes were kept in screened cloth cages (45 cm × 40 cm × 40 cm) with a long cloth sleeve on one side to prevent the escape of any mosquito from the cage. A wet cotton pad was kept on the top of each cage to provide water to the mosquitoes. Deseeded, water-soaked split raisins kept in a Petri plate were placed in the cage, primarily as a source of food for the male mosquitoes. Female mosquitoes were provided with blood meal on alternate days by keeping a restrained albino rat in the cage for at least 2 h during the day. The day following blood meal, an ovitrap consisting of an enamel bowl lined on all sides with the Whatman filter paper and half-filled with dechlorinated water was kept in the cage for the collection of eggs. Dechlorinated water was prepared by storing tap water in large plastic buckets for 24 h. The strips of the filter paper laden with the eggs were taken out on every alternate day and kept moist for 2 days to allow embryonation of eggs. A few strips were subsequently dried and stored in sterilized bottles. The bottles were stocked in the refrigerator at 4°C. The eggs, thus stored, were viable for at least 6 months. Whenever required, the stored egg strips were dipped into deoxygenated water, which was prepared by boiling distilled water in an Erlenmeyer flask and then cooling it without access to the air. This resulted in synchronous and immediate hatching of larvae within 20‒ 25 min.

The remaining egg strips were dipped in the dechlorinated water for maintenance of culture. The newly emerged larvae were transferred in dechlorinated water-filled enamel trays (25 cm × 30 cm × 5 cm) for rearing. Each day, food consisting of finely ground dog biscuits and yeast (3:1 by weight) was provided to the larvae. Paramount care was taken to prevent scum formation on the surface of water. The larval period of *Ae. aegypti* comprising four instars, lasted for 9‒ 10 days. Pupae formed were isolated into an enamel bowl containing water which was then kept in the cloth cage for adult emergence. Blood meal was provided to the female mosquitoes on the third day after emergence.

**• Okhla, New Delhi** (latitude: 28° N 34′ 2.583″, longitude: 77° E 17′ 32.039″, altitude: 214 m)

**• Dwarka, New Delhi** (latitude: 28° N 35′ 31.704″, longitude: 77° E 2′ 45.773″, altitude: 212

**• Faridabad, Haryana** (latitude: 28° N 24′ 32.08″, longitude: 77° E 19′ 4.041″, altitude: 205 m) **• Shahdara, New Delhi** (latitude: 28° N 41′ 10.29″, longitude: 77° E 16′ 29.987″, altitude: 211

**• Noida, Uttar Pradesh** (latitude: 28° N 35′ 12.99″, longitude: 77° E 20′ 27.61″, altitude: 201 m) **• Ghaziabad, Uttar Pradesh** (latitude: 28° N 38′ 11″, longitude: 77° E 25′ 38.1″, altitude: 384

The larvae and adults collected from different locations were brought to the laboratory. The stages were critically observed and those of *Aedes* were segregated and bought back to the

The colony of *Ae. aegypti* was maintained in an insectary under controlled conditions of 28 ± 1°C, 80 ± 5% relative humidity and 14:10 light/ dark photoperiod [30]. Adult mosquitoes were kept in screened cloth cages (45 cm × 40 cm × 40 cm) with a long cloth sleeve on one side to prevent the escape of any mosquito from the cage. A wet cotton pad was kept on the top of each cage to provide water to the mosquitoes. Deseeded, water-soaked split raisins kept in a Petri plate were placed in the cage, primarily as a source of food for the male mosquitoes. Female mosquitoes were provided with blood meal on alternate days by keeping a restrained albino rat in the cage for at least 2 h during the day. The day following blood meal, an ovitrap consisting of an enamel bowl lined on all sides with the Whatman filter paper and half-filled with dechlorinated water was kept in the cage for the collection of eggs. Dechlorinated water was prepared by storing tap water in large plastic buckets for 24 h. The strips of the filter paper laden with the eggs were taken out on every alternate day and kept moist for 2 days to allow embryonation of eggs. A few strips were subsequently dried and stored in sterilized bottles. The bottles were stocked in the refrigerator at 4°C. The eggs, thus stored, were viable for at least 6 months. Whenever required, the stored egg strips were dipped into deoxygenated water, which was prepared by boiling distilled water in an Erlenmeyer flask and then cooling it without access to the air. This resulted in synchronous and immediate hatching of larvae

The remaining egg strips were dipped in the dechlorinated water for maintenance of culture. The newly emerged larvae were transferred in dechlorinated water-filled enamel trays (25 cm × 30 cm × 5 cm) for rearing. Each day, food consisting of finely ground dog biscuits and yeast (3:1 by weight) was provided to the larvae. Paramount care was taken to prevent scum formation on the surface of water. The larval period of *Ae. aegypti* comprising four instars, lasted for 9‒ 10 days. Pupae formed were isolated into an enamel bowl containing water which was then kept in the cloth cage for adult emergence. Blood meal was provided to the female

**3.2. Laboratory colonization and maintenance of** *Ae. aegypti*

**• Alipur, New Delhi** (latitude : 28° N 48′, longitude : 77° E 8′ 59.999″, altitude : 211 m)

m)

138 Current Topics in Chikungunya

m)

m)

laboratory for rearing.

within 20‒ 25 min.

mosquitoes on the third day after emergence.

**Figure 1.** Areas selected in Delhi and NCR for the collection of *Aedes aegypti* larvae:(a) overview; (b) enlarged view.

#### **3.3. Collection of plant material**

The fruits of three citrus plants *C. sinensis*, *C. limon* and *C. limetta* were gathered from the surrounding areas of New Delhi, India in the sterilized polythene bags. The peels were separated from all the three species of fruits and were thoroughly washed with tap water. Care was taken to clean dust, particles or any unwanted material stuck to them. The peels were also observed cautiously to detect any kind of disease or infection. The infected peels were discarded while the selected peels were kept for drying under shade at room temperature (27 ± 2°C). The peels were dried completely ensuring to prevent fungal or bacterial growth.

#### **3.4. Preparation of peel extract**

The dried peels were mechanically grinded using a small blender. The grounded material was sieved to get fine powder. The 15 g of each dried and powdered citrus peels was extracted with 200 ml of hexane and petroleum ether, separately. The extraction was carried out for 8 h per day and continued for consecutive 3 days, using Soxhlet extractor at a temperature not exceeding the boiling point of the solvent. The six extracts obtained were concentrated using a vacuum evaporator at 45°C under low pressure and stored in a refrigerator at 4°C as the stock solution of 1000 ppm for further use.

#### **3.5. Larvicidal bioassay**

The larvicidal bioassay was performed at 28 ± 1°C on the *Ae. aegypti* early fourth instars in agreement with the methodology described by WHO with minor modifications [31]. The graded series of each peel extract was prepared using ethanol as the solvent. The early fourth instars of *Ae. aegypti* were separated, in groups of 20, in plastic bowls filled with 99 ml of distilled water. The larvae were transferred to a glass jar containing 1 ml of the particular concentration of extract added to 100 ml of distilled water. For each dilution, four simultaneous replicates were carried out making a total of 80 larvae for each extract concentration. On the other hand, in controls, ethanol was added to water instead of peel extract. During the exposure period, the larvae were not provided with any food. The dead and moribund larvae were recorded after 24 h as larval mortality.

#### **3.6. Data analysis**

The tests with more than 20% mortality in control assays and 20% pupae formed were rejected and performed again. In case, the control mortality ranged between 5% and 20%, it was rectified using Abbott's formula [32].

$$\text{Corrected mortality} = \frac{\% \text{ Test Mortality} - \% \text{Control mortality}}{100 - \% \text{Control mortality}} \times 100 \%$$

The data was subjected to the regression analysis using computerized SPSS 18.0 Programme. The LC50 and LC90 values with 95% fiducial limits were calculated in each bioassay to measure difference between the test samples. Other statistical parameters, such as standard error and regression coefficient, were also recorded.

#### **3.7. Contact irritancy assays**

**3.3. Collection of plant material**

140 Current Topics in Chikungunya

**3.4. Preparation of peel extract**

**3.5. Larvicidal bioassay**

**3.6. Data analysis**

stock solution of 1000 ppm for further use.

recorded after 24 h as larval mortality.

rectified using Abbott's formula [32].

regression coefficient, were also recorded.

The fruits of three citrus plants *C. sinensis*, *C. limon* and *C. limetta* were gathered from the surrounding areas of New Delhi, India in the sterilized polythene bags. The peels were separated from all the three species of fruits and were thoroughly washed with tap water. Care was taken to clean dust, particles or any unwanted material stuck to them. The peels were also observed cautiously to detect any kind of disease or infection. The infected peels were discarded while the selected peels were kept for drying under shade at room temperature (27 ± 2°C). The peels were dried completely ensuring to prevent fungal or bacterial growth.

The dried peels were mechanically grinded using a small blender. The grounded material was sieved to get fine powder. The 15 g of each dried and powdered citrus peels was extracted with 200 ml of hexane and petroleum ether, separately. The extraction was carried out for 8 h per day and continued for consecutive 3 days, using Soxhlet extractor at a temperature not exceeding the boiling point of the solvent. The six extracts obtained were concentrated using a vacuum evaporator at 45°C under low pressure and stored in a refrigerator at 4°C as the

The larvicidal bioassay was performed at 28 ± 1°C on the *Ae. aegypti* early fourth instars in agreement with the methodology described by WHO with minor modifications [31]. The graded series of each peel extract was prepared using ethanol as the solvent. The early fourth instars of *Ae. aegypti* were separated, in groups of 20, in plastic bowls filled with 99 ml of distilled water. The larvae were transferred to a glass jar containing 1 ml of the particular concentration of extract added to 100 ml of distilled water. For each dilution, four simultaneous replicates were carried out making a total of 80 larvae for each extract concentration. On the other hand, in controls, ethanol was added to water instead of peel extract. During the exposure period, the larvae were not provided with any food. The dead and moribund larvae were

The tests with more than 20% mortality in control assays and 20% pupae formed were rejected and performed again. In case, the control mortality ranged between 5% and 20%, it was

% Test Mortality % Control Mortality Corrected mortality 100

The data was subjected to the regression analysis using computerized SPSS 18.0 Programme. The LC50 and LC90 values with 95% fiducial limits were calculated in each bioassay to measure difference between the test samples. Other statistical parameters, such as standard error and

100 % Control Mortality - = ´ - Whatman filter paper circles were impregnated with 50 ppm hexane and petroleum ether peel extracts of *C. sinensis*, *C. limon* and *C. limetta,* separately. These papers were completely dried and used afresh for contact irritancy assays. Each paper was placed on a glass plate and a Perspex funnel with a hole on the top was kept inverted over them. The 3-day- old unfed female adults of *Ae. aegypti* were separated. Single female was released inside the funnel on the paper. The opening on the top of funnel was plugged with a cotton swab. The adult was left undis‐ turbed to settle for 3 min after which the time taken for the first take-off flight was recorded. The assay was continued for 15 min during which the total number of flights undertaken by that mosquito was scored. Similar parallel control tests were performed with ethanol-impreg‐ nated papers. Each treatment had three replicates. Data was analysed and the relative irritability of the extract in each case was calculated with respect to control.

#### **3.8. Phytochemical analysis**

All the extracts were subjected to preliminary screening of phytochemical investigation and the components in each extract were identified using standard protocols as documented by Harborne et al. [33]. Various qualitative tests were performed to discover the presence of alkaloids, phenolic compounds, carbohydrates, flavonoids, tannins, phlobatannins, saponins and terpenoids in the extracts.

#### **4. Results**

Present investigations were performed with an objective to formulate a safe and environmentfriendly strategy for minimization of waste in fruit juice processing industry by employing citrus fruit peel waste as the larvicidal agent against *Ae. aegypti*. Keeping this in view, the hexane and petroleum ether extracts of *C. Sinensis*, *C. limon* and *C. limetta* peels were assessed for their larvicidal and irritant potential against early fourth instar larvae and non-bleed fed females of *Ae. aegypti*. The comparative result of larvicidal bioassays of peel extracts performed on early fourth instar larvae of *Ae. aegypti* with hexane and petroleum ether extracts of *C*. *sinensis*, *C. limon* and *C. limetta* peels are presented in Tables 2‒ 4, respectively. The results prove and establish the efficacy of all the extracts of citrus peels against the mosquito larvae.

Our investigations showed that the hexane extracts of peels were more effective than petro‐ leum ether extracts irrespective of the citrus species. The hexane extract of *C. sinensis* peels proved to be more effectual larvicidal agent against *Ae. aegypti* resulting in LC50 values as low as 39.51 ppm. Nevertheless, petroleum ether peels extract of *C. limon* was the most effective larvicidal agent against *Ae. aegypti* with LC50 value of 51.25 ppm, while that of *C. limetta* peel wastes showed least activity exhibiting LC50 value of 145.50 ppm. All treatments resulted in complete mortality without any pupa or adult emergence. The control or untreated groups did not show any mortality within 24 h. The larvae developed into pupae and then adults within 48‒72 h (Figure 2).


**Table 2.** Larvicidal activity of extracts prepared from the peels of *Citrus sinensis* against early fourth instars of *Aedes aegypti*


**Table 3.** Larvicidal activity of extracts prepared from the peels of *Citrus limon* against early fourth instars of *Aedes aegypti*


**Table 4.** Larvicidal activity of extracts prepared from the peels of *Citrus limetta* against early fourth instars of *Aedes aegypti*

**Figure 2.** Comparison of LC50 value of hexane and petroleum ether extracts prepared from the peels of *Citrus sinensis*, *Citrus limon* and *Citrus limetta* against early fourth instars of *Aedes aegypti*.

When the females of *Ae. aegypti* were subjected to contact irritancy assays, a significant response was observed (Table 5). The results revealed the more irritant potential of petroleum ether extracts as compared to hexane extracts. The petroleum ether extract of *C. limon* was found to be the most effective against *Ae. aegypti* with first time of flight recorded as 14s with maximum irritability effect of 25-fold (Table 5).


Figures in parentheses indicate standard deviation.

**Extraction solvent**

142 Current Topics in Chikungunya

*aegypti*

Petroleum ether

*aegypti*

**Extraction solvent**

*aegypti*

**Extract LC50**

**LC50 (ppm)**

**(ppm)**

**LC50 (ppm)**

**LC50**

**Concentration**

 **(ppm)**

39.51

55.58

*Citrus limon* and *Citrus limetta* against early fourth instars of *Aedes aegypti*.

**95% Fiducial limits**

**95% Fiducial limits**

> **95% Fiducial limits**

**LC90 (ppm)**

Hexane 39.51 35.22‒43.66 61.90 54.73‒74.99 0.97 2.16 (5) 6.57 Petroleum ether 55.58 50.22‒60.62 85.05 75.56‒104.51 1.15 7.06 (5) 6.93

> **LC90 (ppm)**

Hexane 46.08 40.10‒52.07 85.34 72.83‒109.29 0.68 4.78 (5) 4.78

**LC90 (ppm)**

Hexane 96.15 83.52‒112.11 163.27 139.21–241.30 1.08 7.58 (6) 5.57 Petroleum ether 145.50 118.05‒180.58 371.48 278.83–596.47 0.47 6.52 (4) 3.14

46.08 51.25

**Peels Extracts**

**Figure 2.** Comparison of LC50 value of hexane and petroleum ether extracts prepared from the peels of *Citrus sinensis*,

**Table 2.** Larvicidal activity of extracts prepared from the peels of *Citrus sinensis* against early fourth instars of *Aedes*

51.25 45.37‒57.83 93.50 79.01‒122.97 0.71 3.36 (6) 4.90

**Table 3.** Larvicidal activity of extracts prepared from the peels of *Citrus limon* against early fourth instars of *Aedes*

**Table 4.** Larvicidal activity of extracts prepared from the peels of *Citrus limetta* against early fourth instars of *Aedes*

96.15

145.5

**95% Fiducial limits**

**95% Fiducial limits**

> **95% Fiducial limits**

**S.E.** *χ<sup>2</sup>*

**S.E.** *χ<sup>2</sup>*

**S.E.** *χ<sup>2</sup>*

 *(df)* **Regression**

 *(df)* **Regression coefficient**

 *(df)* **Regression**

*Citrus sinensis (Hexane)*

*Citrus sinensis (PE)*

*Citrus limon (Hexane)*

*Citrus limon (PE)*

*Citrus limetta (Hexane)*

*Citrus limetta (PE)*

**coefficient**

**coefficient**

**Table 5.** Response of of non-blood fed females of *Ae. aegypti* to papers impregnated with extracts of *Citrus sinensis*, *Citrus limon* and *Citrus limetta* in the contact irritancy assays


**Table 6.** Phytochemical screening of *Citrus* peels

The preliminary qualitative phytochemical analysis of the peel extracts of all the three *Citrus* sp. discovered the occurrence of terpenoids and flavonoids in both the hexane and petroleum ether extracts (Table 6). Other components, i.e., carbohydrates, phenolic compounds, saponins, tannins and phlobatannins were not noticed in any of these extracts. Nevertheless, the petroleum ether extract exhibited alkaloids, the presence of which was not observed in the hexane extracts.

#### **5. Discussion**

It is well documented that the control of mosquito-borne diseases can be achieved either at adult stage preventing mosquitoes to bite human beings by using repellents/irritants or at larval stage causing mortality at a large scale in the breeding areas. Nonetheless, the wide‐ spread use of synthetic insecticides has caused environmental risks and the development of insecticide resistance in the vector species. The adverse impact of insecticides has caused concern and necessitated the search and development of environmentally safe, biodegradable, economically effective and indigenous methods for mosquito control, which can be employed with least care by human beings and communities [1].

A number of reports documented in the field of mosquito control reveal the worthiness of diverse phytochemicals procured from various plant species against different mosquito species. Sukumar et al. made an extensive review of botanical derivatives which have been investigated against mosquitoes [10]. They reported a large number of plant extracts which possess cidal or repellent activities against mosquito vectors, but indicated that very few plant products have shown practical value for mosquito control. Plants are known to be rich sources of complex mixtures of bioactive compounds that can be used to develop environmentally safe vector and pest-managing agents. Nevertheless, natural pesticides derived from plants are a promising tool especially for targeting mosquitoes in larval stage [21]. Our experiments reveal the hexane extracts of the peels of all the three *Citrus* species were highly effective against *Ae. aegypti* as compared to petroleum ether extracts.

Very few reports are available regarding larvicidal and contact irritancy of citrus peels against *Ae. aegypti.* Our results are in agreement with that of Murugan et al. who had tested the efficacy of *C. sinensis* ethanolic peel extract against *Ae. aegypti* and *Cx. quinquefasciatus* and found it to be more effective against *Ae. aegypti* having LC50 value of 92.27 ppm whereas against *Cx. quinquefasciatus* the value reported was 244.70 ppm [34]. The essential oils extracted from peels of *C. limon* and *C. sinensis* have been reported to exhibit strong toxicity against larvae of *Cx. pipiens* with the LC50 values ranging from 30.1 to 51.5 mg/ l [24]. Akram et al. had found extracts prepared from the seeds of rough lemon and lemon as effective larvicides with LC50 values of 119.993 and 137.258 ppm, respectively, after 24 h of exposure and 108.85 and 119.853 ppm, respectively, after 48 h of exposure [35]. The volatile peel extracts of *C. sinensis* and *C. aurantifolia* (lime) were also reported to have insecticidal activity against mosquitoes [25].

Our studies also showed significant irritant potential of petroleum ether extract of *C. limon* against *Ae. aegypti* as compared to *C. sinensis* and *C. limetta* extracts. Similar repellency behaviour in *Ae. aegypti* was reported by Kumar et al. when adults were exposed to the leaf extracts of *Parthenium hysterophorus* prepared in different solvents [36]. Essential oils from a few verbenaceae plants have also shown repellent activity against *Ae. aegypti*, *An. stephensi* and *Cx. quinquefasciatus* [17]. Similarly repellency of aromatic plants and pure components was studied by Abdallah et al. against *Cx. pipiens molestus* [37]. The potential of volatile oils extracted from turmeric, citronella grass and hairy basil as topical repellents have also been reported against *Ae. aegypti*, *An. dirus* and *Cx. quinquefasciatus* [38]. Thangam also studied repellent activity of acetonic extracts of four seaweeds *Caulerpa peltata*, *C. racemosa*, *C. scalpel‐ liformis* and *Diclyota dichotoma* and eleven mangrove plant samples, *Avicennia marina*, *A. officinalis*, *Excoecaria agallocha*, *Lumnitzera racemosa*, *Rhizophora apiculata*, *R. lamarckii*, *R. mucro‐ nata*, *Salicornia brachiata*, *Sonneratia apetala*, *Xylocarpus granatum*, against *Ae. aegypti* out of which he found the stilt root of *Rhizophora apiculata* extract to be most effective [39]. Petroleum ether extract of *Vitex negundo* leaves offered bite protection for 8 h (2 mg/cm2 ) by different mosquitoes in the field [40]. This suggests that natural pesticides derived from plants are a promising tool for mosquito control programs.

Earlier studies have showed that phytochemicals play a major role in mosquito control programme. The secondary metabolites of plants (such as steroids, alkaloids, terpenoids, saponins, phenolics, essential oil, etc.) are associated with a wide range of biological activities. Marston et al. have reported the efficacy of prenylated xanthones, tetracyclic phenols and saponins in controlling *Ae. aegypti* [26]. Likewise, the presence of carbohydrates, saponins, phytosterols, phenols, flavonoids and tannins have been observed in the plant extract exhib‐ iting mosquito larvicidal activity [27]. Furthermore, the citrus fruit peel has been found to be a rich source of flavanones and many polymethoxylated flavones, rare in other plants [28].

The present study showed the presence of flavonoids and terpenoids as the common constit‐ uents in the hexane and petroleum ether extracts of *Citrus* sp. These results are in conformity with that of Kumar et al. who showed the presence of alkaloids, flavonoids and terpenoids in the petroleum ether extracts of *C. sinensis* peels [29]. The presence of flavonoids and cardiac glycosides in methanol extract of *Lantana camara* leaves and flowers, flavonoids in leaf and terpenoids in the flower of ethanol extract has been shown by Sathish and Maneemegalai [41]. Rawani et al. suggested that the presence of bioactive principles such as steroids, alkaloids, terpenes, saponins, etc. may be responsible for the larvicidal properties of crude extracts of three plants, *viz. Carica papaya*, *Murraya paniculata* and *Cleistanthus collinus* against *Cx. quin‐ quefasciatus* [42]. Vinayachandra et al. concluded that either the presence of saponins, phenol‐ ics, steroids or terpenoids in the plant extracts of *Knema attenuata* or a combination of two or more of these metabolites might be the cause for larvicidal efficacy against the Indian strains of *An. stephensi* and *Ae. albopictus* [43]. Although our investigations have revealed the occur‐ rence of only two phytochemical constituents, the larvicidal potential of the extracts might be because of the synergistic effects of other phytochemical constituents present in the extracts, identified or unidentified in the present investigation.

#### **6. Conclusion**

tannins and phlobatannins were not noticed in any of these extracts. Nevertheless, the petroleum ether extract exhibited alkaloids, the presence of which was not observed in the

It is well documented that the control of mosquito-borne diseases can be achieved either at adult stage preventing mosquitoes to bite human beings by using repellents/irritants or at larval stage causing mortality at a large scale in the breeding areas. Nonetheless, the wide‐ spread use of synthetic insecticides has caused environmental risks and the development of insecticide resistance in the vector species. The adverse impact of insecticides has caused concern and necessitated the search and development of environmentally safe, biodegradable, economically effective and indigenous methods for mosquito control, which can be employed

A number of reports documented in the field of mosquito control reveal the worthiness of diverse phytochemicals procured from various plant species against different mosquito species. Sukumar et al. made an extensive review of botanical derivatives which have been investigated against mosquitoes [10]. They reported a large number of plant extracts which possess cidal or repellent activities against mosquito vectors, but indicated that very few plant products have shown practical value for mosquito control. Plants are known to be rich sources of complex mixtures of bioactive compounds that can be used to develop environmentally safe vector and pest-managing agents. Nevertheless, natural pesticides derived from plants are a promising tool especially for targeting mosquitoes in larval stage [21]. Our experiments reveal the hexane extracts of the peels of all the three *Citrus* species were highly effective against *Ae.*

Very few reports are available regarding larvicidal and contact irritancy of citrus peels against *Ae. aegypti.* Our results are in agreement with that of Murugan et al. who had tested the efficacy of *C. sinensis* ethanolic peel extract against *Ae. aegypti* and *Cx. quinquefasciatus* and found it to be more effective against *Ae. aegypti* having LC50 value of 92.27 ppm whereas against *Cx. quinquefasciatus* the value reported was 244.70 ppm [34]. The essential oils extracted from peels of *C. limon* and *C. sinensis* have been reported to exhibit strong toxicity against larvae of *Cx. pipiens* with the LC50 values ranging from 30.1 to 51.5 mg/ l [24]. Akram et al. had found extracts prepared from the seeds of rough lemon and lemon as effective larvicides with LC50 values of 119.993 and 137.258 ppm, respectively, after 24 h of exposure and 108.85 and 119.853 ppm, respectively, after 48 h of exposure [35]. The volatile peel extracts of *C. sinensis* and *C. aurantifolia* (lime) were also reported to have insecticidal activity against mosquitoes [25].

Our studies also showed significant irritant potential of petroleum ether extract of *C. limon* against *Ae. aegypti* as compared to *C. sinensis* and *C. limetta* extracts. Similar repellency behaviour in *Ae. aegypti* was reported by Kumar et al. when adults were exposed to the leaf extracts of *Parthenium hysterophorus* prepared in different solvents [36]. Essential oils from a few verbenaceae plants have also shown repellent activity against *Ae. aegypti*, *An. stephensi* and

with least care by human beings and communities [1].

*aegypti* as compared to petroleum ether extracts.

hexane extracts.

144 Current Topics in Chikungunya

**5. Discussion**

Our studies have clearly demonstrated that waste peels of citrus fruits can be utilized as the effective agents of mosquito control. The utilization of wastes as beneficial products would not only assist to reduce waste load by managing intractable waste discharge but would also diminish the pollution load and improve the environmental profile of fruit juice processing industry.

#### **Acknowledgements**

The authors are grateful to Dr Savithri Singh, Principal, Acharya Narendra Dev College, for providing the laboratory and culture facilities to conduct the experimental work.

### **Author details**

Sarita Kumar\* , Monika Mishra, Aarti Sharma and Radhika Warikoo

\*Address all correspondence to: saritakumar@andc.du.ac.in

Department of Zoology, Acharya Narendra Dev College, University of Delhi, Delhi, India

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not only assist to reduce waste load by managing intractable waste discharge but would also diminish the pollution load and improve the environmental profile of fruit juice processing

The authors are grateful to Dr Savithri Singh, Principal, Acharya Narendra Dev College, for

providing the laboratory and culture facilities to conduct the experimental work.

, Monika Mishra, Aarti Sharma and Radhika Warikoo

Department of Zoology, Acharya Narendra Dev College, University of Delhi, Delhi, India

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148 Current Topics in Chikungunya


## *Edited by Alfonso J. Rodriguez-Morales*

Chikungunya, an arbovirus, is a major global threat affecting multiple areas of the world, even Europe, but recently (2014 - 2015) with large epidemics in Latin America, causing an important acute and chronic morbidity with a low, but present, mortality. This book tries to update the significant epidemiological and clinical research in many aspects with a multinational perspective. This book has been organized in two major sections: (I) ''Clinical and Epidemiological Aspects'' and (II) ''Entomology.'' Section I includes topics covering experiences and studies in different countries, including the infection during pregnancy and children, imported cases, ocular manifestations, coinfections, and therapeutics. Section II includes topics on entomological aspects, related to vector control, and new options for biological control of Aedes aegypti.

Photo by Hans Verburg / iStock

Current Topics in Chikungunya

Current Topics in

Chikungunya

*Edited by Alfonso J. Rodriguez-Morales*