This chapter focuses on the method of controlling juvenile mosquitoes while in life stages (larvae & pupae) which only occur in water. To safely alter our aquatic environments, even temporarily, for the purpose of controlling mosquitoes, requires a good working knowledge of both the target species and larvicides, which include commercial pesticides and natural predators. Products and techniques currently used in Florida are discussed in detail, and the benefits and risks of each are considered. Commercial pesticide information includes summaries and information provided by manufacturers. Minor differences between various formulations of the same or similar active ingredients are detailed so that the competency of each product may be compared. The old days of smothering everything with one pesticide such as waste oil are gone, and mosquito control is rapidly approaching an age of prescription applications where a competent operator will apply one or a combination of larvicides in an environmentally friendly manner under a given set of conditions.

Commercial pesticide sections summarize data found in manufacturers’ current product literature and labels. Two of many additional sources of information on mosquito larvicides are:

• U.S. Environmental Protection Agency HTTP://WWW.EPA.GOV/PESTICIDES/HEALTH/MOSQUITOES/LARVICIDES4MOSQUITOES.HTM
• Alameda County, California Mosquito Control HTTP://WWW.MOSQUITOES.ORG/BIORAT.HTML
The University of Florida published a handbook (Dean and Nesheim 1998) on correct pesticide applications which covers in depth many topics presented here.

5.1 INTRODUCTION
Larviciding is a general term for killing immature mosquitoes by applying agents, collectively called larvicides, to control mosquito larvae and/or pupae. Larval Source Management (LSM) involves both the modification of water habitats, often referred to as Source Reduction (see Chapter 4), and the direct application of larvicides to control mosquito production. Most mosquito species spend much of their life cycle in the larval

stage when they are highly susceptible to both predation (see Chapter 7) and control efforts. They often are concentrated within defined water boundaries, immobile with little ability to disperse, and accessible. Adult mosquitoes, in contrast, fly in search of mates, blood meals, or water sources for egg laying and are often inaccessible, not concentrated, and widely distributed. Therefore, effective larviciding can reduce the number of adult mosquitoes available to disperse, potentially spread disease, create a nuisance, and lay eggs which leads to more mosquitoes.

The effective control of larvae and/or pupae is a basic principle of Integrated Pest Management (IPM). Effective IPM involves understanding the local mosquito ecology and patterns of arbovirus transmission and then selecting the appropriate mosquito control tools. The most common methods of IPM include Environmental Management, or Source Reduction (Chapter 4), Larviciding, and Adulticiding (Chapter 6). Other mosquito control principles include Biocontrol (Chapter 7), as well as additional methods not discussed here such as herbiciding and hand removal of aquatic plants. These methods may be used to control immature mosquitoes indirectly, usually when there is an obligatory association between the larvae/pupae and specific host plants. In Florida, Mansonia and Coquellittidia mosquitoes are associated with aquatic plants.

Common examples of highly concentrated broods include immature Aedes taeniorhynchus and Ae. sollicitans in saltmarsh pools, Psorophora columbiae in flooded pastures, and species such as Culex nigripalpus in wastewater treatment sites. In these situations, most Florida mosquito control programs larvicide as a management practice because it both minimizes the area in which control procedures must be applied and reduces the need for adult control. At these times, larviciding has a high impact on local population numbers with minimal application efforts. At other times, larviciding may be less rewarding because small numbers of larvae and pupae are widely and unevenly distributed. Examples include Culiseta melanura in bay tree swamps, Mansonia species and Cq. perturbans in large freshwater marshes with patchy host plant distribution, and Anopheles quadrimaculatus in large, overgrown grassy retention ponds.

Planning a LSM strategy is crucial to a highly effective control program. The first step begins with adult and larval surveillance. Once surveys have been conducted, it is then important to map out and prioritize potential larval habitats. Treatment thresholds, often based on the number of larvae encountered at a site, should be established to justify larviciding, and action plans appropriate for the sites should be developed.

It is important to select the appropriate control agent and formulation based on performance and other factors. It is critical to have a thorough knowledge of the biology of the targeted species in order to determine the appropriate larvicide, the timing of the application, and the amount of product to be applied. For example, Ae. taeniorhynchus tend to “ball up” when feeding as 3rd instars (Nayar 1985). The larvae are unevenly distributed and the density where they do occur is much higher than at other times in their development when they tend to be more evenly dispersed in salt marsh pools. This situation may call for an application rate higher what is normally used, but never exceeding the maximum allowed on the label. Larvicides may be chosen which exhibit a selective mode of action and have a minimal residual activity or which are not selective and exhibit long-term control. Many larvicides can be applied from either the ground by truck, boat, and hand held devices or by air with fixed wing and rotary wing aircraft, however, some products are not suitable for aerial application. Follow-up efficacy checks are important to ensure a successful larviciding program, and rotation of products should be incorporated into any IPM program.

There is no perfect larvicide for every situation, and each larvicide has its strengths and weaknesses. Larvicides may be grouped into two broad categories: biorational pesticides and conventional, broad-spectrum pesticides. The latter will be discussed in sections 5.2.3 thru 5.2.4.2.

The term “biorational” gained popularity in the climate of environmental awareness and public concern (Williamson 1999). It refers to pesticides of natural origin that have limited or no adverse effects on the environment or beneficial organisms. In order for a synthetically produced pesticide to be classified as a biorational, it must be structurally identical to a naturally occurring compound. Biorational pesticides are comprised of two major categories: 1) Microbial agents (e.g., bacteria) HTTP://WWW.PESTICIDEBOOK.COM/PDFS/CHAPTER24_PAGES293-295.PDF and 2) Biochemical agents (e.g., pheromones, hormones, growth regulators, and enzymes).

Schuster and Stansly (2006) more recently defined a biorational pesticide as any type of insecticide active against pest populations but relatively innocuous to non-target organisms, and, therefore, non-disruptive to biological control. An insecticide can be "innocuous" by having low or no direct toxicity on non-target organisms or by having short field residual, thereby minimizing exposure of natural enemies to the insecticide. By this definition, all larvicides registered for use in Florida, when applied according to label instructions, might be considered biorational. There is actually no legally clear, absolute definition of a biorational pesticide (Williamson 1999). The U.S. Environmental Protection Agency (EPA) considers biorational pesticides to have different modes of action than traditional pesticides (HTTP://IPMWORLD.UMN.EDU/CHAPTERS/WARE.HTM), with greater selectivity and considerably lower risks to humans, wildlife, and the environment. The EPA lists several larval control agents as “biopesticides” (HTTP://WWW.EPA.GOV/OPPBPPD1/BIOPESTICIDES/INGREDIENTS/INDEX.HTM). The terms “biorational” and “biopesticide” overlap but are not identical.

5.1.1 HISTORY
Stories of prodigious numbers of mosquitoes occupy a special place in Florida’s history (Patterson 2004) beginning with 16th century explorers. An 1888 yellow fever epidemic in Jacksonville set in motion the formation of the Florida State Board of Health (FSBH) in 1889. The Florida Anti-Mosquito Association was founded in 1922. The first mosquito control legislation was passed, and the Indian River Mosquito Control District was established in 1925 (Anonymous 1948, Patterson 2004).

Larviciding became prominent when implemented as an area-wide malaria control procedure in the early 1900s, but by then it had been used as a control technique for over a century in Florida (Floore 2006). From the earliest days, two types of larval control were employed: Larviciding as a temporary control method and ditching as a permanent control method (see Chapter 4 on Source Reduction). Larviciding using waste oil or diesel oil products was implemented to control mosquitoes in the early 1800s (Howard 1910). Paris green dust, an arsenical insecticide, was developed as a larvicide in 1865 and, along with undiluted diesel oil, was used through the 1960s (Anonymous 1970). In 1958, the FSBH developed its own Paris-green granular formulation as a general purpose larvicide (Mulrennan 1958). The FSBH went on to develop its own “Florida Mosquito Larvicide” in the 1960s which contained 99% mineral oil (unpublished 24-C label 1967).

After 1945, dichloro-diphenyl-trichloroethane (DDT), a chlorinated hydrocarbon compound, was used as both an adulticide and a larvicide in Florida (Anonymous 1970, Patterson 2004). Mosquitoes became resistant to DDT, and its use was discontinued in the late 1950s. As resistance to DDT increased, malathion, an organophosphate (OP) compound, was used increasingly to control both larval and adult mosquitoes. Soon, resistance to malathion was observed in saltmarsh mosquitoes (Rathburn and Boike 1967). The FSBH then implemented a policy limiting the use of malathion to adulticiding in areas where OP larvicides were not used. Resistance (see Chapter 10) has been a concern of Florida mosquito control agencies (Boike and Rathburn 1968) for many years. Rogers and Rathburn (1964) summarized early agency attitudes toward larviciding: “Although larviciding alone is not regarded as a practical procedure for mosquito control in Florida … the great value of larvicides is fully appreciated.” Attitudes have changed, and by 2006 most mosquito control agencies in Florida had incorporated larviciding as one of their mosquito management practices.

During the ten years that have elapsed since the first edition of this document, a number of larviciding formulations are no longer registered and likely will never again be available as tools for mosquito control agencies. These products include pyrethrum, diflubenzuron, Bonide Mosquito Larvicide (oil), and BVA Chrysalin (oil). Laginex AS (active ingredient Lagenidium giganteum) has not been enthusiastically accepted in Florida or elsewhere in the United States. Some agencies may list predatory minnows which they purchase for larval control as line items in their larvicide budgets, but these fish are considered biocontrol agents. Biocontrol is discussed in Chapter 7. Industry consolidation has placed the stewardship of the remaining larvicides into the hands of fewer manufacturers. Mosquito control professionals must be diligent with applications and guard against the loss of the remaining control agents.

5.1.2 REGULATION
The regulation of larvicides and larviciding is provided for by a set of federal and state acts, statutes, and rules. Oversight includes both regulation of the pesticides themselves and regulation of pesticide applications. The principal controlling law is the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). Chapter 487 Florida Statutes (F.S.), known as “The Florida Pesticide Law”, Chapter 388 F.S. known as “The Mosquito Control Act” and associated Rules outlined in Chapters 5E-2 and 5E-13 of the Florida Administrative Code constitute the State’s authority (HTTP://WWW.FLAES.ORG/AESENT/INDEX.HTML). The Florida Department of Agriculture and Consumer Services (FDACS), Bureau of Entomology and Pest Control, is tasked with ensuring compliance and regulates and licenses the pest control industry and mosquito control programs.

In accordance with FIFRA and the Florida Pesticide Law, FDACS has established a Pesticide Review Council (PRC) to advise “the Commissioner of Agriculture regarding the sale, use and registration of pesticides and advises government agencies, including the State University System, regarding their responsibilities pertaining to pesticides” (HTTP://WWW.FLAES.ORG/PESTICIDE/PESTICIDEREVIEWCOUNCIL.HTML). The Council serves as a statewide forum for the coordination of pesticide related activities to eliminate duplication of effort and maximize protection of human health and the environment. The PRC consists of eleven scientific members and operates under the authority of Chapter 487 F.S.

The FDACS Division of Agricultural Environmental Services (AES) administers various state and federal regulatory programs concerning environmental and consumer protection issues. These responsibilities include state mosquito control program coordination, agricultural pesticide registration, testing, and regulation, pest control regulation, and feed, seed, and fertilizer production inspection and testing. The AES, Bureau of Pesticides, Pesticide Registration Section “registers federally accepted (FIFRA) pesticides” (HTTP://WWW.FLAES.ORG/PESTICIDE/PESTICIDEREGISTRATION.HTML) that are distributed, sold, or offered for sale in Florida. Pesticides not requiring federal approval must be registered in Florida to assure adherence with State law. Emergency exemptions from federal registration also are reviewed and processed by the Pesticide Registration Section and submitted to the EPA for action. Special registration actions for new active ingredients, special local needs, significant new uses, and experimental use permits are processed through the Section. To accomplish their mission, members of the Section consult with specialists within FDACS and other state and federal agencies, commissions, and councils.

The Scientific Evaluation Section (SES) of the FDACS, AES, Bureau of Pesticides, includes scientists with expertise in geology, soil science, hydrology, mammalian and ecological toxicology, chemistry, and chemical fate modeling (HTTP://WWW.FLAES.ORG/PESTICIDE/SCIENTIFICEVALUATION.HTML). The SES provides technical support and has five core functions/programs:

1. Pesticide Registration Evaluation Committee Reviews
2. Endangered Species Protection Program
3. Ground Water Protection Program
4. Surface Water Protection Program
5. Pesticide Usage Information Reporting
The SES functions and interacts with other stakeholders to ensure the safety of the State of Florida. Many new mosquito control insecticide formulations are evaluated by the Florida Agricultural and Mechanical University, John A. Mulrennan, Sr. Public Health Entomology Research and Education Center (PHEREC) in Panama City.

Chapter 388 F.S. provides the authority for mosquito control activities. The statute includes a provision that public lands may be designated as environmentally sensitive and biologically highly productive, thereby requiring special arthropod control plans for mosquito control activities on those “designated” lands. Many state and federal land management authorities [e.g., Florida Department of Environmental Protection (FDEP), Florida Division of Forestry (FDOF), Florida Fish and Wildlife Conservation Commission (FFWCC), U.S. Fish and Wildlife Service (USFWS)] and regional water management districts designate their conservation lands similarly and have corresponding control plans in place.

The control plans are initially proposed by the mosquito control agency for individual parcels and negotiated with the public land manager until mutually agreed upon. Either party may propose further amendments. There is no overarching agreement that certain control chemicals are approved for all such public lands. For example, in 1987, the Florida Park Service and various mosquito control agencies adopted control plans for many state parks (personal communication, Dana C. Bryan, Environmental Policy Coordinator, Office of the Director, Florida Park Service, December 2006). At that time, products containing Bacillus thuringiensis israelensis (Bti) and methoprene were widely approved for use. Bacillus sphaericus (Bs) had not yet been developed commercially and hence was not included in arthropod control plans. Many subsequent plans include Bs in addition to Bti and methoprene. See Chapters 9 and 13 for additional discussions of mosquito control agency interactions with other government entities.

5.2 LARVICIDES AVAILABLE
Mosquito larvicides registered for use in Florida are discussed below within the following classification system:

1. Insect growth regulators (IGRs)
2. Microbial larvicides • organophosphates (OPs)
3. Organophosphates (OPs)
4. Surface oils and films
Insecticide labels usually bear a precautionary signal word. The necessity for a signal word on labels (HTTP://WWW.EPA.GOV/OPPFEAD1/LABELING/LRM/CHAP-07.HTM) and which word is assigned is dependent upon the results of six separate acute toxicity studies which are performed with each product formulation.

There are a variety of products and formulations within each larvicide classification. Specific formulations are different from manufacturer to manufacturer. Application rates and suggested treatment sites may differ as well.Individual product labels and material safety data sheets (MSDS), usually downloadable from manufacturers’ web sites, should

be consulted for specific information, habitat dependent application rates, and restrictions, if any. FDACS should be consulted to ensure that a specific product is labeled for use in Florida.

5.2.1 INSECT GROWTH REGULATORS (IGRS)
The initial identification of a natural juvenile hormone (JH I) in insects occurred in 1967 and was followed rapidly by the discovery of JH II and JH III (Henrick 2007). JH is involved in the regulation of physiological processes in insects including mating and metamorphosis. Research was initiated in 1968 to determine if insect pests could be selectively controlled – without environmental concerns – by developing synthetic mimics of the natural JH. Since JH does not occur in vertebrates, it was expected that selective insecticides could be developed. Sacher (1971) reported on a group of chemicals that mimic juvenile hormone activity. These chemicals appeared to block naturally occurring ecdysone from initiating molting processes and inducing metamorphosis in mosquito larvae. Staal (1975) discussed several methoprene analogs that interfere with normal insect growth and maturation. Abnormal larval growth patterns plus malformed or smaller than normal forms were observed. The first IGR, which contained several methoprene isomers, was registered in 1975 (Henrick 2007). Methoprene products currently are the only IGRs registered for use in Florida.

5.2.1.1 METHOPRENE
Methoprene (Isopropyl (2E, 4E, 7S)-11-methoxy -3,7,11 -trimethyl-2,4-dodecadienoate) is a terpenoid compound. Technical methoprene is an amber or pale yellow liquid with a faint fruity odor (HTTP://EXTOXNET.ORST.EDU/PIPS/METHOPRE.HTM), which is slightly soluble in water and is miscible in organic solvents. Methoprene is a synthetic mimic and a true analog of naturally occurring JH found in mosquitoes and in other insects.

JH is found throughout the larval stages of a mosquito, but it is most prevalent during the early instars. As mosquito larvae mature, the level of naturally occurring JH steadily declines until just prior to the 4th instar molt, when larvae develop into pupae. This time is a sensitive period when all the physical features of the adult begin to form. Methoprene is absorbed through the insect’s outer "skin" or cuticle and may be incidentally ingested or enter the body through other routes. The level of applied methoprene (parts per billion) in the larvae’s water environment must be higher than the level of juvenile hormone circulating in the larvae’s body in order for the disruption of endocrine processes to occur. Therefore, the application of methoprene larvicides is most efficacious during late 4th instar. Treated larvae reach the pupal stage and then cannot emerge to become adults. Since pupae do not eat, they eventually deplete body stores of essential nutrients and starve to death. Incomplete adult emergence is an indicator of methoprene efficacy.

Methoprene is listed (HTTP://WWW.EPA.GOV/OPPBPPD1/BIOPESTICIDES/INGREDIENTS/INDEX.HTM by the EPA as a biopesticide. Methoprene based larvicides are General Use Pesticides (GUPs). Methoprene-based larvicides have undergone extensive studies both prior to and after registration to determine risk to humans and non-target organisms. When used according to label directions, methoprene is considered extraordinarily safe for humans and almost all non-target organisms. Methoprene does not produce nondiscriminatory, rapid toxic effects often associated with central nervous system toxicants. The lethal effects of methoprene are based on the disruption of the insect’s endocrine system mediated developmental processes, such as metamorphosis and embryogenesis. Consequently, control of mosquito larvae is relatively slow.

Methoprene is effective in a wide variety of both fresh and saltwater habitats. It is relatively selective for target species, and lingering mosquito pupae serve as a food for fish and other predators. The IGR is particularly effective against Aedes larvae. Methoprene does not bioaccumulate; it degrades into simpler compounds. Since ultraviolet light deactivates methoprene, many formulations incorporate activated charcoal or other dark inert substances to prolong product life. Early methoprene manufacturing products included two mirror-image molecules called r- and s-isomers. The racemic isomer (r-methoprene) is not active on mosquitoes. Improved manufacturing techniques allow current formulations to contain only active s-methoprene isomers. Methoprene labels bear the “CAUTION” signal word.

5.2.2 MICROBIAL LARVICIDES
Microbial larvicides are formulated to deliver a natural toxin to the intended target organisms. Bacteria are single-celled parasitic or saprophytic microorganisms that exhibit both plant and animal properties and range from harmless and beneficial to intensely virulent and lethal. Bacillus thuringiensis (Bt), is the most widely used agricultural microbial pesticide in the world, and the majority of microbial pesticides registered with the EPA are based on Bt. The Bt serovar kurstaki (Btk) is the most commonly registered microbial pesticide, and this variety has activity against Lepidoptera (butterflies and moths) larvae. It was originally isolated from natural Lepidopteran die-offs in Germany and Japan. Bt products have been available since the 1950s. In the 1960s and 1970s, the World Health Organization (WHO) encouraged and subsidized scientific discovery and utilization of naturally occurring microbes. As a result of those early studies and a whole body of subsequent work, two lines of mosquito control products have been developed: crystalline toxins of two closely related gram-positive, aerobic bacteria – Bacillus thuringiensis israelensis (Bti) and Bs. Mosquito control agents based on Bt are the second most widely registered group of microbial pesticides. Highly successful Bti products have expanded the role of microbial agents into the public health arena (de Barjac 1990). Reviews of microbial agents may be found in Lacey 1985, Lacey 2007, and Singer 1985.

5.2.2.1 BACILLUS THURINGIENSIS ISRAELENSIS
Bacillus thuringiensis is a bacterium which occurs naturally in soils and aquatic environments globally. In 1976, Goldberg and Margalit (1977) isolated Bti from Culex pipiens collected in an Israeli riverbed. In 1977, de Barjac designated this Bt strain as H14, noting that it is toxic to mosquito and black fly larvae. Over the last three decades, a number of other strains have been investigated, some with desired larvicidal effects. Two strains, SA3A and FM65-52, are currently utilized for commercial products.

The active ingredients in Bti formulations are delta-endotoxin (d-endotoxin) crystals separated from bacteria near the end of manufacturing processes. These toxic crystals are incorporated into various products which allow their release into water so that they may be ingested by mosquito larvae. The d-endotoxin crystals are activated by the alkaline environment and enzymes of the mosquito midgut. The alkaline gut environment allows hydrolysis of the crystal’s protein coating and the release of pro-toxins. Gut enzymes then activate the pro-toxins and facilitate their binding to the gut epithelium of the mosquito larva. Cells rupture and are destroyed at the binding sites, leading to a loss of body fluids which results in death. This rapid action typically controls larvae in 4-24 hours.

Copyright.http://mosquito.ifas.ufl.edu/Larviciding.htm