OF PESTS IN FARMED FISH ECOSYSTEMS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/381,367, filed October 28, 2022, and entitled “COMPOSITIONS AND METHODS FOR THE MANAGEMENT OF PESTS IN FARMED FISH ECOSYSTEMS,” which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The present disclosure relates to the management or prevention of pest infections or infestations fish ecosystems in and around farmed fish sea cages. In specific, the disclosure provides compositions and methods for reducing, preventing, or controlling ectoparasitic infections or infestations in farmed fish ecosystems by administering pest control agents to both farmed fish and wild fish present within the farmed fish ecosystems.

BACKGROUND

[0003] Aquaculture of various fish species provides a critical food source for a growing world population. In 2021, it was estimated that about 175 million metric tons of fish were produced for global consumption. An increase in demand for fish by consumers has resulted in an increase in the number of aquaculture farms around the world. With an increase in aquaculture farms comes an increased need for sustainable technologies for safe and effective pest control in farmed fish populations.

[0004] Many pests exist that are a threat to fish raised in aquaculture systems. For example, farmed fish raised in captivity are susceptible to various parasitic infections and infestations, including from ectoparasites and endoparasites. If left uncontrolled, parasitic infections and infestations can cause disease, developmental delays, and malformities in the fish. Such infections and infestations have significant impacts on the health and growth of the fish and can lead to significant production and economic losses for farmers. Parasitic infection often weakens the host fish, which makes them susceptible and more likely to suffer detrimental effects from other causes beyond the initial damage from the parasite. In particular, various types of farmed fish, such as farmed Atlantic salmon (Salmo salctr . are prone to infections and infestations with a number of species of ectoparasites, an example being the salmon louse Lepeophtheirus salmonis. Similar types of sea lice infect various other types of fish throughout the world, including throughout the Pacific Ocean along the coast of Asia. These types of parasites can have significant impacts on the marketability of farmed fish due to the negative impacts the parasites have on the appearance of the fish, including marks, blemishes, and sores on the skin and in the muscle of the fish. The wounds caused by feeding activities can lead to reduced growth, osmoregulatory failure, and even mortality in small fish. Sea lice can exert an immunomodulatory effect on their fish hosts and as a result they can evade any innate or adaptive immunity of the host. Further, fish stressed and immunocompromised by lice experience disruptions in wound healing and can become predisposed to secondary microbial infections. Finally, sea lice present in farmed fish populations produce a large number of eggs that can get carried by water currents into the open sea thus posing a risk to wild salmonid populations and neighboring fish farms. Some countries, such as Norway, regulate the maximum allowable number of adult female lice on salmon to from 0.2 to 0.5 female lice per fish, putting additional pressures on farmers to control the pests.

[0005] Controlling parasites costs the global aquaculture industry an estimated $750 million USD a year due to the need to purchase agents such as parasiticides and expensive equipment, lost productivity from starvation and underfeeding associated with physical delousing systems, excess mortality due to physical delousing systems, and the further need to invest in personnel time for the management, control, and research of new parasite management and control methods. In many cases, farmers must monitor their fish to keep sea lice within pre-determined thresholds set by each region, and when necessary, they must cull fish to prevent suffering and the spread of the parasites, further contributing to economic losses. The use of various first generation parasiticides, including chemotherapeutants such as azamethiphos, cypermethrin, deltamethrin, diflubenzuron, emamectin benzoate, and hydrogen peroxide, have been shown to have negative effects on the fish after long-term use. Some negative effects include a failure of the fish to grow to their full size due to various reasons, including fasting before or after the physical delousing systems, refusal to ingest various agents, and reduced appetite. Further, the use of such antiparasitic agents has caused concern by consumers about resistance and the potential for residual antiparasitic agents left in the flesh of the fish or in the environment.

[0006] While fish raised in open sea cages are particularly susceptible to such parasitic infections and infestations due to the flow of water into and out of the open sea cages, fish raised in fully enclosed sea cages or semi-enclosed sea cages are also susceptible to such parasitic infections and infestations. Various circumstances can lead to fish raised in fully enclosed sea cages or semi-enclosed sea cages becoming infected or infested with sea lice ectoparasites in farmed fish ecosystems. For example, overtopping of water in the sea cages with open top sides, failures of water filtration systems, or accidental inclusion of sea lice during the addition of new smolt into the sea cages.

[0007] In addition, farmed fish are not the only type of fish afflicted by parasitic infections and infestations in the farmed fish ecosystem. It is common for wild fish to become infected or infested with the same parasitic organisms as farmed fish when the wild fish enter into the farmed fish ecosystem. A significant number of wild fish live in and around the farmed fish ecosystem, and during various times, such as during a post smolt salmon migration, these wild fish pass in close proximity to the farmed fish sea cages where they can come into contact with various parasites. As the wild fish pass through the farmed fish ecosystem they can acquire a parasitic infection or infestation along their migration path. While various regulatory agencies lower the regulatory limits of 0.5 adult female sea lice per fish to 0.2 adult female sea lice per fish during migration periods in order to protect the migrating smolt that pass by stationary sea cages to lessen the chance of infection, infections still result in the wild fish populations.

[0008] Thus, there exists a need to control various parasitic infections and infestations in farmed fish ecosystems, including those parasitic infections and infestations found in farmed fish populations in open and enclosed (e.g., fully enclosed or semi-enclosed) sea cages, as well as in wild fish populations within the farmed fish ecosystem.

SUMMARY

[0009] The present disclosure provides a method for reducing, preventing, or controlling a parasitic infection or infestation in a farmed fish ecosystem including administering to farmed fish in one or more enclosed or semi-enclosed sea cages a fish feed including a pest control agent, the pest control agent including a neem extract rich in azadirachtin A.

[0010] In an aspect, the method further includes where neem extract rich in azadirachtin A includes from 15 wt. % to 33 wt. % azadirachtin A.

[0011] In an aspect, the method further includes where fish feed is administered upon introduction of fish into the enclosed sea cages.

[0012] In an aspect, the method further includes where fish feed is administered for a period of time prior to introduction of fish into the enclosed sea cages.

[0013] In an aspect, the method further includes where enclosed sea cages are fully enclosed sea cages or semi-enclosed sea cages. [0014] In an aspect, the method further includes where fish feed is administered to the fish for at least 14 days.

[0015] In an aspect, the method further includes where neem extract rich in azadirachtin A is administered to the fish at a concentration from 1.5 mg to 2.5 mg azadirachtin A per kg body weight per day.

[0016] In an aspect, the method further includes where neem extract rich in azadirachtin A is administered to the fish at a concentration from 2.6 mg to 5.0 mg azadirachtin A per kg body weight per day.

[0017] In an aspect, the method further includes administering a second fish feed including a pest control agent including a neem extract rich in azadirachtin A to wild fish within the farmed fish ecosystem.

[0018] In an aspect, the method further includes where the parasitic infection or infestation is an ectoparasitic infection or infestation, or an endoparasitic infection or infestation.

[0019] In an aspect, the method further includes where the parasitic infection or infestation is a sea lice infection or infestation, or a copepod infection or infestation.

[0020] In an aspect, the method further includes where the parasitic infection or infestation includes an infection or infestation with a copepod including one or more species of Caligus or Lepeophtheirus.

[0021] In an aspect, the method further includes where the parasitic infection or infestation includes an infection or infestation with a copepod including one or more of Caligus clemensi, Caligus elongatus, Caligus rogercresseyi, or Lepeophtheirus salmonis.

[0022] In an aspect, the method further includes administering a pest control agent composition to the farmed fish before, during, or after administration of the fish feed.

[0023] In an aspect, the method further includes where the neem extract does not include neem oil.

[0024] In an aspect, the method further includes where the neem extract rich in azadirachtin A is obtained by a method including the steps of providing neem seeds; crushing the neem seeds; extracting azadirachtin from the crushed seeds with water; adding a second extraction solution that includes a non-aqueous solvent which is not miscible with water and has a higher solubility of azadirachtin than water or a surfactant having a turbidity temperature between 20 °C and 80 °C; and recovering the concentrated azadirachtin from the second extraction solution. [0025] In an aspect, the method further includes where the fish feed further includes one or more components including antibiotic agents, antibacterial agents, antifungal agents, antiviral agents, antiparasitic agents, or antiprotozoal agents.

[0026] In an aspect, the method further includes where the fish feed is administered to species of farmed fish belonging to one or more families including Cyprinidae, Cichlidae, Pangasiidae, Sciaenidae, Serranidae, Carangidae, Sparidae, Lateolabracidae, Moronidae, Mugilidae, Cypriniformes, Latidae, Eleotridae, Tilapiini, and Salmonidae.

[0027] The present disclosure provides a method for reducing, preventing, or controlling a parasitic infection or infestation in a farmed fish ecosystem including administering to wild fish present in the farmed fish ecosystem a first fish feed including a pest control agent, the pest control agent including a neem extract rich in azadirachtin A.

[0028] In an aspect, the method further includes where the neem extract rich in azadirachtin A includes from 15 wt. % to 33 wt. % azadirachtin A.

[0029] In an aspect, the method further includes administering to farmed fish within the farmed fish ecosystem a second fish feed including a pest control agent, the pest control agent including a neem extract rich in azadirachtin A.

[0030] In an aspect, the method further includes administering the second fish feed to the farmed fish at the same time as administering the first fish feed to the wild fish.

[0031] In an aspect, the method further includes administering the second fish feed to the farmed fish at a different time than administering the first fish feed to the wild fish.

[0032] In an aspect, the method further includes where the neem extract rich in azadirachtin A in the first fish feed is administered to the wild fish at the same concentration as the neem extract rich in azadirachtin A in the second fish feed that is administered to the farmed fish.

[0033] In an aspect, the method further includes where the first fish feed is administered to the wild fish having a different concentration of neem extract rich in azadirachtin A than the neem extract rich in azadirachtin A in the second fish feed that is administered to the farmed fish.

[0034] In an aspect, the method further includes where the first fish feed and the second fish feed are administered at a concentration of azadirachtin A from about 0.001 wt. % to 10.0 wt. % azadirachtin A.

[0035] In an aspect, the method further includes where the parasitic infection or infestation is an ectoparasitic infection or infestation, or an endoparasitic infection or infestation.

[0036] In an aspect, the method further includes where the parasitic infection or infestation is a sea lice. [0037] In an aspect, the method further includes where the neem extract rich in azadirachtin A does not include neem oil.

[0038] In an aspect, the method further includes where the fish feed further includes one or more components including antibiotic agents, antibacterial agents, antifungal agents, antiviral agents, antiparasitic agents, or antiprotozoal agents.

[0039] In an aspect, the method further includes where the fish feed is administered to any species of wild fish belonging to one or more families including Cyprinidae; Cichlidae; Pangasiidae; Sciaenidae; Serranidae; Carangidae; Sparidae; Lateolabracidae; Moronidae; Mugilidae; Cypriniformes; Latidae; Eleotridae; Tilapiini and Salmonidae.

[0040] In an aspect, the method further includes where the parasitic infection includes an infection or infestation with a copepod including one or more species of Caligus or Lepeophtheirus.

[0041] In an aspect, the method further includes where the ectoparasitic infection includes an infection or infestation with a copepod including one or more of Caligus clemensi, Caligus elongatus, Caligus rogercresseyi, or Lepeophtheirus salmonis.

[0042] In an aspect, the method further includes administering a pest control agent composition including a neem extract rich in azadirachtin A to the farmed fish or the wild fish before, during, or after administration of the first fish feed.

[0043] The present disclosure provides a neem extract rich in azadirachtin A for reducing, preventing, or controlling a parasitic infection or infestation in a farmed fish ecosystem by following the steps including administering to farmed fish in one or more enclosed or semienclosed sea cages a fish feed including the neem extract rich in azadirachtin A.

BRIEF DESCRIPTION OF THE FIGURES

[0044] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed herein. Aspects may be more completely understood in reference to the following drawings, in which:

[0045] FIG. 1 is a schematic diagram showing the lifecycle of the salmon louse Lepeophtheirus salmonis in accordance with various aspects herein.

[0046] FIG. 2 is a schematic plot diagram of exemplary development of sea lice present in a fish population as a function of time and various targeted management models. DETAILED DESCRIPTION

[0047] Reference will now be made in detail to certain aspects of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0048] As described above, both farmed fish and wild fish face many challenges from infection and infestation caused by pests present within a farmed fish ecosystem. Efforts to address such infections and infestations, such as exposing the fish populations to first-generation chemical agents in open sea cages using either bath exposures or in-feed exposures have also presented many challenges. These types of techniques can result in toxicity to the fish, increased resistance to the chemical agents by the parasites, persistence in the environment, and increased scrutiny by consumers. Bath exposures can include the administration of drugs dissolved in water through complete immersion for a pre-determined period. In-feed exposures can include mixing one or more active agents with a fish feed or coating the active agents onto a fish feed. An advantage of bath exposures in well boats is that the parasites are exposed to the same concentration of the drug. Nevertheless, bath exposures can be very labor intensive, the transfer of fish to well boats causes losses of livestock, the pest control agents used in the well boats can be toxic and polluting to the environment, and the active ingredient in the pest control agents may degrade before it reaches all of the target pests.

[0049] In-feed agent delivery can be less stressful both for the fish and for the farmers and can permit the simultaneous administration of agent to most or all of the fish cages regardless of the weather conditions, and thus reduce the risk of cross-infection with adjacent cages. Further, various physical delousing systems also exist to manage such infections and infestations, but the mechanical and thermal technologies used in such systems can be damaging to the fish and can have limited efficiency. Fish farmed in enclosed sea cages can be susceptible to accidental exposure to parasites when prevention measures fail in the enclosed systems. Attempts at controlling or preventing parasitic infections or infestations in farmed fish generally address the infections or infestations experienced by the farmed fish, however, the increased concentration of parasites within the farmed fish ecosystem has significant impacts on the wild fish populations as well. The present disclosure provides compositions and methods for managing pest infections and infestations in various farmed fish ecosystems through the use of one or more pest control agents, one or more semiochemical compounds, one or more pest control agent compositions, or one or more fish feeds as described herein.

[0050] As used herein, the term “infection” can refer to a condition where a pestilent organism, including the various pests defined elsewhere herein, can invade any internal or external portion of a host organism’s body such that the host organism experiences harm, and where the pestilent organism uses components of the host organism to sustain itself, reproduce, or colonize the host organism.

[0051] As used herein, the term “infestation” can refer to the presence of an abnormally large number of pests as defined herein, where the pests are concentrated in a region in numbers that can cause damage or disease through infection of a host organism.

[0052] As used herein, the term “pest” can refer to any organism that is detrimental to the health, value, or appearance of another organism. The term pest can include, but is not to be limited to, one or more of various parasites including worms, helminths, flukes, lice, mites; one or more species of bacteria; one or more viruses; one or more type of fungi; and various protozoa (e.g., amoeba).

[0053] As used herein, the term “parasite” can refer to one or more species of ectoparasite or endoparasite. As used herein, the term “endoparasite” can refer to organisms that inhabit one or more internal niches of another organism. For example, an endoparasite can inhabit one or more of the tissues, organs, or systems of a host organism. For example an endoparasite can inhabit the gut, blood, or both, of a host organism. As used herein, the term “ectoparasite” can refer to organisms that inhabit or occupy an external niche of another species. For example, an ectoparasite can inhabit or occupy the surface of a host species. In the case of fish, ectoparasites can inhabit the skin of the fish where they sometimes lodge between scales, and they further can feed off of the mucus, blood, skin, gills, muscle, or any combination thereof. Ectoparasites can include species belonging to the phylum Arthropoda. As such, the term ectoparasites further can include crustaceans and one or more species of sea lice that inhabit fish hosts. In an aspect, the ectoparasites can include one or more species of copepod.

[0054] As used herein, the term “pest control agent” can refer to an agent for reducing, preventing, or controlling an infection or infestation caused or contributed to by one or more pests. In various aspects, the pest control agents described herein can refer to an agent for reducing, preventing, or controlling an infection or infestation caused by one or more endoparasites or ectoparasites. [0055] As used herein, the term “farmed fish ecosystem” refers to a population of farmed fish and any other organisms found within the physical environment of the farmed fish, including any other organisms found within open sea cages, enclosed sea cages, and the physical environment in which the open sea cages or enclosed sea cages are disposed. The other organisms found within the farmed fish ecosystem can include microorganisms, macroorganisms, plants, fungi, protozoa, and the like. It will be appreciated that the other organisms can encompass one or more types of wild fish and various pests as described elsewhere herein. It will be appreciated that any water flowing into and out of a farmed fish ecosystem at any given time is a part of the physical environment of the farmed fish ecosystem at that time.

[0056] As used herein, the term “semiochemical compound” refers to one or more behaviormodifying chemicals produced by an organism that can affect the behavior of that same species of organism or the behavior of a different species of organism. A semiochemical compound can include one or more types of pheromones, allomones, kairomones, synomones, any of which can function as either an attractant or repellant, or both.

Fish Feeds

[0057] The present disclosure provides fish feeds to be used as fish diets in aquaculture applications. It will be appreciated that the fish feeds herein can include suitable types of fish feed specific for a given fish species. The fish feeds can be used as a component of a diet fed to any species belonging to the families Cyprinidae, Cichlidae, Pangasiidae, Sciaenidae, Serranidae, Carangidae, Sparidae, Lateolabracidae, Moronidae, Mugilidae, Cypriniformes, Latidae, Eleotridae, Tilapiini and Salmonidae. In various aspects, this disclosure provides a fish feed or fish feed diet for species within the family Salmonidae. The fish feeds provided herein can be used to feed wild fish or farmed fish. In various aspects, both wild fish and farmed fish can be fed simultaneously. Further, the fish feed can be used to feed freshwater fish or salt water (e.g., marine) fish, or both.

[0058] The fish feeds of the present disclosure can be produced using a base feed formulation that is a solid feed or a liquid feed using raw materials that can be chosen based on the application in which it is to be used and on the fish species. In various aspects, the fish feed is a solid fish feed. In other aspects, the fish feed is a liquid fish feed. In other aspects, the fish feed can include both a solid fish feed component and a liquid fish feed component. Fish feeds in solid form can include pellets, extruded nuggets, steam pellets, flakes, tablets, powders, and the like. In various aspects, the base feed can include a base feed pellet. In some aspects, the base feed pellet can include a porous matrix distributed throughout. Fish feeds in liquid form can include aqueous solutions, oils, oil and water emulsions, slurries, suspensions, and the like. In various aspects, a solid fish feed can further include one or more oils disposed on the surface or distributed throughout the fish feed.

[0059] The fish feeds herein can include a number of different ingredients or raw materials that can sustain life, growth, and reproduction of the fish. The fish feeds can include any substrate that is edible to fish. For example, an edible substrate can provide a source of nutrition to the fish or can be an inert substrate with no nutritive value to the fish. In various aspects, the fish feeds herein can include feeds that are either nutritional fish feeds or non-nutritional feeds. Nutritional fish feeds can include a nutritional food stuff formulated for fish as part of its diet as the main source of nutrition, growth, and reproduction. Suitable nutritional fish feeds can include one or more of proteinaceous material as a source of proteins, peptides, and amino acids; carbohydrates; and fats, as described below. Non-nutritional fish feeds can include any substrate that is edible to fish but does not provide nutrition to sustain life, growth, or reproduction. In various aspects, the nutritional or non-nutritional fish feeds herein can include one or more compounds designed to alter the quality, quantity, or appearance of a fish and fish tissue. For example, a nutritional or non-nutritional fish feed can include a carotenoid compound to improve the appearance (e.g., color) of the muscle tissue. By way of example, the carotenoid compound can include compounds such as astaxanthin.

[0060] The fish feeds herein can include a complete fish feed. A complete fish feed can include a feed for fish that is compounded to be fed as the sole ration and that can maintain life, promote growth, and sustain reproduction without any additional substances being consumed except water. Complete fish feeds can include compounded mixtures containing various energy sources such as carbohydrates, proteins, and fats. In various aspects, the fish feeds herein can include at least a protein and a starch. Additional ingredients can be included, such as vitamins and minerals as necessary to support the life, growth, and reproduction of fish. A complete fish feed can include ingredients such as, but not limited to, fish meal, poultry meal, plant meal, vegetable meal, corn meal, corn gluten meal, soy meal, soy protein concentrate, single cell protein, insect meal, algae meal, algae oil, krill meal, krill oil, meat meal, blood meal, feather meal, starches, tapioca starch, wheat, wheat gluten, guar meal, guar protein concentrate, peas, pea protein concentrate, pea starch, beans, faba beans, sunflower meal, vegetable oil, canola oil, poultry oil, rapeseed oil, fish oil, soy oil, linseed oil, camelina oil, lecithin, macro-minerals, minerals, vitamins, amino acids, pigments, and any combinations thereof. It will be appreciated that the fish feeds herein can include fish meal that can include plant or animal derived matter. Any animal derived matter present in the fish meal can be derived from the same species of fish or a different species of fish (e.g., heterologous species or non-fish species).

[0061] The total protein content in the fish feed can be from 10 wt. % to 70 wt.%, from 15 wt.% to 65 wt.%, from 20 wt.% to 60 wt. %, or from 25 wt.% to about 55 wt.%. The total protein in the fish feed can be at least 10%, 15%, 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 55%, 60%, 65%, or at least 70% by weight, or any amount within a range of any of the forgoing. The total protein in the fish feed can be variable depending on the formulation, species, age, and intended use of the feed. It will be appreciated that the various protein requirements of the species of fish receiving the fish feed can be adjusted to meet the protein requirements of that species.

[0062] The protein in the fish feeds herein can be from any suitable source including, but not limited to, one or more of fish meal, land-animal protein (e.g., poultry meal), plant-based protein (e.g., vegetable meal), or any combinations thereof. The fish feed can include from 0% to 80%, from 10% to 80%, from 20% to 75%, from 30% to 70%, from 60% to 80%, or from 10% to 30%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 75% fish meal by weight, or any amount within a range of any of the forgoing. The fish feed can include from 0% to 80%, from 10% to 80%, from 20% to 75%, from 30% to 70%, from 60% to 80%, or from 10% to 30%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 75% land-animal protein by weight, or any amount within a range of any of the forgoing. The fish feed can include between 0% to 80%, from 10% to 80%, from 20% to 75%, from 30% to 70%, from 60% to 80%, or from 10% to 30%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 75% plant-based protein by weight, or any amount within a range of any of the forgoing.

[0063] Total fat (e.g., oil, fat, and/or lipids) in the fish feed can be from 5% to 50%, from 10% to 45%, from 15% to 40%, or from 20% to 35%. The total fat in the fish feed can be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50% by weight, or any amount within a range of any of the forgoing. The total fat in the fish feed can be variable depending on the formulation, target fish species, and intended use of the fish feed. It will be appreciated that the various fat requirements of the species of fish receiving the fish feed and can be adjusted to meet the fat requirements of that species. Suitable fats for use herein can include, but are not to be limited to, those provided by canola oil, poultry oil, rapeseed oil, fish oil, soy oil, linseed oil, camelina oil, palm oil, lecithin, or any combinations or fractions thereof. [0064] The moisture content of the fish feeds herein can vary depending on the contents and preparation method of the feed. In various aspects, the moisture content can be from 1% to 20%, from 2% to 18%, from 5% to 15%, or from 6% to 12% by weight.

[0065] The fish feeds herein can include one or more pest control agents. In various aspects, the one or more pest control agents can be present in the fish feed in an amount effective to produce an inhibitory effect on one or more pests, as will be described elsewhere herein. As such, the fish feeds herein can include pest control agents for controlling infections or infestations caused by one or more pests. In various aspects, the fish feeds herein can include pest control agents for controlling endoparasitic or ectoparasitic infections or infestations. In various aspects, the parasitic infection or infestation is a copepod infection or infestation. In some aspect, the parasitic infection or infestation is a sea lice infection or infestation. Various pests suitable as targets for the pest control agents herein are described elsewhere.

[0066] Each pest control agent included within the fish feeds herein can be individually capable of controlling one or more of a parasitic, bacterial, viral, fungal, or protozoal infections or infestations. Therefore, it should be understood that any given pest control agent for use in the many aspects described herein can be referred to as exhibiting one or more inhibitory effects, including antiparasitic effects (e.g., anti-ectoparasitic, anti-endoparasitic), antibacterial effects, antiviral effects, antifungal effects, or antiprotozoal effects. In various aspects, the inhibitory effect can include an antiparasitic effect, where the antiparasitic effect can further include an anti- ectoparasitic effect, an anti-endoparasitic effect, or both. It will be appreciated that the inhibitory effects can result in reducing, preventing, or controlling the concentration and spread of the various parasitic, bacterial, viral, fungal, or protozoal organisms described herein. In various aspects, a pest control agent of the present disclosure can produce inhibitory effects against one or more pests including one or more effects for reducing, preventing, or controlling the concentration and spread of various endoparasites or ectoparasites. Reducing, preventing, or controlling the parasites can include complete prevention of infection or infestation in the fish population or on each fish, a reduction in the total number of parasites present in the fish population or on each fish, or controlling how many parasites are present in the fish population or on each fish according to local regulatory requirements. It will be appreciated that the inhibitory effects described herein can be measured against a population fish infected or infested with one or more pests that are fed a diet lacking the neem extract rich in azadirachtin A.

[0067] In other aspects, the inhibitory effect against the pests can include one or more of an anti-feedancy effect, an anti-molting effect, an antifertility and anti-fecundity effect, or an antiparasitic effect. As used herein, “anti-feedancy effect” can refer to an effect exerted by one or more pest control agents that stops or inhibits feeding by the pests resulting in their malnourishment, delayed development, prevention or delay of molting, and death. Malnourished sea lice are also less likely to efficiently immunomodulate their hosts, and thus they are less capable evading host immunity. As used herein, the term “anti-molting” can refer to an effect exerted by one or more pest control agents that prevents or delays the process of molting in the pests. The process of molting occurs as pests grow and shed their exoskeletons from one life stage to the next and is controlled hormonally and neuronally, and the pest control agents herein can exert one or more anti-molting effects against the pests. As used herein, the terms “antifertility effect” and “anti-fecundity effect” are referred to collectively as an “antifertility and anti-fecundity effect” and can include one or more effects on male or female reproduction. It will be appreciated that the term “fertility” can refer to the actual number of offspring born to or eggs released from a female, and the term “fecundity” can refer to the biological potential for reproduction, and due to their close relationship the two terms as used herein can be used interchangeably unless otherwise noted. An antifertility and anti -fecundity effect can include a reduction in total gamete production in males and females, a complete or partial inhibition of viable egg production, a change in the anatomy and morphology of the gametes of males or females, a change in the potential for egg fertilization, and a reduction in the total number of gravid female pests.

[0068] In an administered form, the fish feeds herein can include an amount of pest control agent at from about 0.01 - 100 grams of pest control agent per kilogram fish feed (g/kg), about 90 g/kg fish feed, about 80 g/kg fish feed, about 70 g/kg fish feed, about 60 g/kg fish feed, about 50 g/kg fish feed, about 40 g/kg fish feed, about 30 g/kg fish feed, about 20 g/kg fish feed, about 1- 10 g/kg fish feed, about 2-9 g/kg fish feed, about 3-7 g/kg fish feed, about 4-6 g/kg fish feed, or about 5 g/kg fish feed.

[0069] In various aspects, the fish feeds herein can include an amount of pest control agent and/or active ingredient in an amount effective to produce an inhibitory effect against one or more pests, including a concentration from about 0.01 g/kg fish feed, 0.05 g/kg fish feed, 0.1 g/kg fish feed, 0.2 g/kg fish feed, 0.3 g/kg fish feed, 0.4 g/kg fish feed, 0.5 g/kg fish feed, 0.6 g/kg fish feed, 0.7 g/kg fish feed, 0.8 g/kg fish feed, 0.9 g/kg fish feed, 1.0 g/kg fish feed. 1.25 g/kg fish feed, 1.5 g/kg fish feed, 1.75 g/kg fish feed, 2.0 g/kg fish feed, 2.25 g/kg fish feed, 2.5 g/kg fish feed, 2.75, g/kg fish feed, 3.0 g/kg fish feed, 5.0 g/kg fish feed. 5.25 g/kg fish feed, 5.5 g/kg fish feed, 5.75 g/kg fish feed, 6.0 g/kg fish feed, 6.25 g/kg fish feed, 6.5 g/kg fish feed, 6.75, g/kg fish feed, 7.0 g/kg fish feed, 7.0 g/kg fish feed. 7.25 g/kg fish feed, 7.5 g/kg fish feed, 7.75 g/kg fish feed, 8.0 g/kg fish feed, 8.25 g/kg fish feed, 8.5 g/kg fish feed, 8.75, g/kg fish feed, 9.0 g/kg fish feed, 9.25 g/kg fish feed, 9.5 g/kg fish feed, 9.75, g/kg fish feed, 10.0 g/kg fish feed, 15 g/kg fish feed, 20 g/kg fish feed, 25 g/kg fish feed, 30 g/kg fish feed, 35 g/kg fish feed, 40 g/kg fish feed, 45 g/kg fish feed, 50 g/kg fish feed, 55 g/kg fish feed, 60 g/kg fish feed, 65 g/kg fish feed, 70 g/kg fish feed, 75 g/kg fish feed, 80 g/kg fish feed, 85 g/kg fish feed, 90 g/kg fish feed, 95 g/kg fish feed, or 100 g/kg fish feed, or any amount within a range of any of the forgoing concentrations.

[0070] It will be appreciated that the aforementioned concentrations equate to an amount from about 0.001-10 weight percent (% w/w) total pest control agent to fish feed. In various aspects, the fish feeds herein can include an amount of pest control agent effective to produce an inhibitory effect against one or more pests including from 0.001 % w/w, 0.002 % w/w, 0.003 % w/w, 0.004 % w/w, 0.005 % w/w, 0.006 % w/w, 0.007 %w/w, 0.008 % w/w, 0.009 % w/w, 0.010 % w/w, 0.020 % w/w, 0.030 % w/w, 0.040 % w/w, 0.050 % w/w, 0.060 % w/w, 0.070 % w/w, 0.080 % w/w, 0.090 % w/w, 0.10 % w/w, 0.11 % w/w, 0.12 % w/w, 0.13 % w/w, 0.14 % w/w, 0.15 % w/w, 0.16 % w/w, 0.17 % w/w, 0.18 % w/w, 0.19 % w/w, 0.20 % w/w, 0.21 % w/w, 0.22 % w/w, 0.23 % w/w, 0.24 % w/w, 0.25 % w/w, 0.26 % w/w, 0.27 % w/w, 0.28 % w/w, 0.29 % w/w, 0.30 % w/w, 0.31 % w/w, 0.32 % w/w, 0.33 % w/w, 0.34 % w/w, 0.35 % w/w, 0.36 % w/w, 0.37 % w/w, 0.38 % w/w, 0.39 % w/w, 0.40 % w/w, 0.41 % w/w, 0.42 % w/w, 0.43 % w/w, 0.44 % w/w, 0.45 % w/w, 0.46 % w/w, 0.47 % w/w, 0.48 % w/w, 0.49 % w/w, 0.50 % w/w, 0.51 % w/w, 0.52 % w/w, 0.53 % w/w, 0.54 % w/w, 0.55 % w/w, 0.56 % w/w, 0.57 % w/w, 0.58 % w/w, 0.59 % w/w, 0.60 % w/w, 0.61 % w/w, 0.62 % w/w, 0.63 % w/w, 0.64 % w/w, 0.65 % w/w, 0.66 % w/w, 0.67 % w/w, 0.68 % w/w, 0.69 % w/w, 0.70 % w/w, 0.71 % w/w, 0.72 % w/w, 0.73 % w/w, 0.74 % w/w, 0.75 % w/w, 0.76 % w/w, 0.77 % w/w, 0.78 % w/w, 0.79 % w/w, 0.80 % w/w, 0.81 % w/w, 0.82 % w/w, 0.83 % w/w, 0.84 % w/w, 0.85 % w/w, 0.86 % w/w, 0.87 % w/w, 0.88 % w/w, 0.89 % w/w, 0.90 % w/w, 0.91 % w/w, 0.92 % w/w, 0.93 % w/w, 0.94 % w/w, 0.95 % w/w, 0.96 % w/w, 0.97 % w/w, 0.98 % w/w, 0.99 % w/w, 1.0 % w/w, 2.0 % w/w, 3.0 % w/w, 4.0 % w/w, 5.0 % w/w, 6.0 % w/w, 7.0 % w/w, 8.0 % w/w, 9.0 % w/w, or 10.0 % w/w, or any amount within a range of any of the forgoing values.

[0071] In various aspects herein, the fish feeds can be administered to the fish having a concentration of pest control agent selected from the group including 0.05 % w/w, 0.06 % w/w, 0.07 % w/w, 0.08 % w/w, 0.09 % w/w, 0.10 % w/w, 0.11 % w/w, 0.12 % w/w, 0.13 % w/w, 0.14 % w/w, 0.15 % w/w, 0.16 % w/w, 0.17 % w/w, 0.18 % w/w, 0.19 % w/w, 0.20 % w/w, 0.30 % w/w, 0.40 % w/w, 0.50 % w/w, 0.60 % w/w, 0.70 % w/w, 0.80 % w/w, 0.90 % w/w, or 1.0 % w/w, pest control agent to fish feed or a range within any of the forgoing concentrations. [0072] In various aspects herein, the fish feeds can include one or more semiochemical compounds as an ingredient. The semiochemical compounds can include one or more semiochemicals that can attract one or more species of wild fish to within the farmed fish ecosystem. The semiochemical compounds can include one or more semiochemicals that can attract one or more species of wild fish to the farmed fish ecosystem to encourage the wild fish migrating past the farmed fish ecosystem to consume the fish feeds. By encouraging the wild fish to consume the fish feeds herein, the potential for pest infection or infestation in the wild fish can be reduced, controlled, or prevented. For example, the semiochemical compounds can include one or more types of pheromones, allomones, kairomones, synomones, of any combination thereof, where any of the semiochemical compounds can function as either an attractant or repellant, or both. In some aspects, the semiochemical compounds can include a combination of one or more types of pheromones, allomones, kairomones, synomones known to attract wild fish to a farmed fish ecosystem. In various aspects, the semiochemical compounds can include wild salmonid pheromones.

Target Fish and Pests

[0073] The fish feeds provided herein can be fed as a fish feed diet or used to feed any fish that is susceptible to infection or infestation by one or more pest. For example, the fish feed can be used in aquaculture as a component of a diet fed to any farmed fish including, for example, commercially relevant fish species. For example, the fish feeds provided herein can form part of diet fed to any of freshwater fish, brackish fish, or saltwater fish. The fish feeds can be used as a component of a diet fed to any species belonging to the families Cyprinidae, Cichlidae, Pangasiidae, Sciaenidae, Serranidae, Carangidae, Sparidae, Lateolabracidae, Moronidae, Mugilidae, Cypriniformes, Latidae, Eleotridae, Tilapiini and Salmonidae. As such, the fish feeds herein can be used to feed species belonging to any of the genera within these families and in particular, those species that are farmed for human or animal consumption. For example, and without limitation, the fish feeds described herein can be used to feed species belonging to the genera Salmo and/or Oncorhynchus . In particular, the fish feeds herein can be used to control pests in populations of wild or farmed salmon or trout species, including, for example, any of Atlantic salmon (Salmo salary Pacific salmon, Char, or Rainbow trout. Moreover, the fish feed can be used as a pest control agent for other fish species within the aquaculture industry such as sea bass, bream, grouper, pompano, and tuna, as well as in the pet and decorative fish industries, for example for pest control in goldfish (Carassius auratus). [0074] Pests of the target fish herein can include ectoparasites including species belonging to the phylum Arthropoda. Susceptible arthropods include various copepods that include many species of sea lice that inhabit fish hosts. By way of example, in the case of fish belonging to the family Salmonidae (e.g., Salmo and/or Oncorhynchus spp.), the pest control agent of the fish feed provided herein can control sea lice infections, sea lice infestations, copepod infections, copepod infestations, or any combinations thereof. The pest control agent present in the fish feeds provided herein can control ectoparasites including parasitic crustaceans, also referred to as copepods, belonging to Argulus ssp. or Caligus ssp. In particular, the pest control agent for use in the fish feed described herein can be effective at controlling copepod infections and infestations of one or more types of farmed fish. As used herein, the term “copepod” refers to a group of crustaceans found in fresh water and in seawater, and which have one or more parasitic phases of their life cycle. Unless otherwise noted, the term copepod can refer to any of the various species of sea lice as described herein.

[0075] A species of copepod that is an ectoparasite of Atlantic salmon belongs to the Lepeophtheirus genus and is known as the salmon louse, Lepeophtheirus salmonis. As used herein, it will be appreciated that the term “sea louse” refers to the singular form and the term “sea lice” refers to the plural form. Each term can be used interchangeably unless otherwise noted. Lepeophtheirus salmonis are a species of copepod ectoparasites that primarily live on salmon, including Atlantic and Pacific salmon, and sometimes on sea trout. Other types of copepod ectoparasites infecting fish belonging to the family Salmonidae include Caligus clemensi, Caligus elongatus, and Caligus rogercresseyi. Sea lice, including those from the genera Lepeophtheirus and Caligus, are ectoparasites which feed off the blood, mucus, muscle, and skin of various salmon species.

[0076] Referring now to FIG. 1, the life cycle of Lepeophtheirus salmonis (i.e., L. salmonis) is shown in accordance with various aspects herein. Various aspects of FIG. 1 have been adapted from Sea Lice Research Centre, 2020, "SLRC - Life cycle of the salmon louse (Lepeophtheirus salmonis ", https://doi.org/10.18710/GQTYYL, DataverseNO, VI. The life cycle of L. salmonis is complex and consists of eight distinct life stages. The nauplius stage begins upon egg hatching and includes two distinct stages, including the nauplius 1 stage and the nauplius 2 stage, shown as 100 and 102, respectively. The nauplius 1 stage 100 and nauplius 2 stage 102 ofZ. salmonis drift passively in water where, in general, both stages are about 0.5 mm to 0.7 mm in length and they are relatively translucent in color. The nauplius 1 stage 100 and nauplius 2 stage 102 exist for about 52 hours and 175 hours, respectively, in temperatures of up to about 15 °C. Upon molting from the nauplius 2 stage 102, L. salmonis transitions into the infective copepodid stage 104 at approximately 10 days of age, with the understanding that development at any life stage can depend on a number of environmental factors, including but not to be limited to temperature, salinity, light hours, and the like. L. salmonis in the copepodid stage 104 are approximately from 0.7 mm to 1.0 mm in length. In the copepodid stage 104, L. salmonis generally attaches to fish along its fins or scales. After attachment, L. salmonis further molts to the chalimus 1 stage 106. In the chalimus 1 stage 106, L. salmonis attaches to the host fish more firmly by way of a frontal filament. The chalimus 1 life stage typically lasts approximately 10 days. The L. salmonis life cycle continues with a molting from chalimus 1 stage 106 to the chalimus 2 stage 108 while remaining firmly attached to the fish. During the chalimus 1 stage 106 and the chalimus 2 stage 108, the L. salmonis is an average length from 1.0 mm to 2.5 mm. The chalimus 2 life stage typically lasts approximately 10 days.

[0077] Following both chalimus stages, L. salmonis molts further into the pre-adult stage where L. salmonis becomes mobile and able to swim or move around the fish surface. At the preadult stage, sexual development begins to differentiate between the females and males. The preadult stage consists of 2 distinct stages, where males develop from chalimus 2 stage 108 into the pre-adult 1 male stage 110 and females develop into the pre-adult 1 female stage 112. The females spend approximately 10 days as the pre-adult 1 female stage 112 and spend approximately 12 days at the pre-adult 2 female stage 116. The males spend approximately 8 days as the pre-adult 1 male stage 110 and spend approximately 9 days at the pre-adult 2 male stage 114. From the pre-adult 1 stages, the sexes continue through development into the pre-adult 2 male stage 114 and the pre- adult 2 female stage 116. During the pre-adult stages, L. salmonis grows to an average length of about 2.5 mm to 3.5 mm. In the final adult stage, the adult male 118 and adult female 120 are distinguishable by size and phenotypic characteristics. Namely, the adult female 120 is from 8 mm to 20 mm in length, including two egg strings 122 visible off the posterior aspect of the organism. In contrast, the adult male 118 is from 5 mm to 7 mm in length.

[0078] On average, L. salmonis can live for approximately up to 215 days for a full life cycle. It will be appreciated that each stage of the L. salmonis life cycle can be dependent on temperature, salinity of the water, water currents, pollution levels, and various additional environmental factors. Thus, the complete life cycle of L. salmonis can be from 32 days up to 215 days depending on the fluctuations in such external factors.

[0079] Thus, in various aspects, the disclosure herein provides a fish feed including a pest control agent capable of any of reducing, preventing, or controlling Lepeophtheirus or Caligus infections or infestations. A Lepeophtheirus infection or infestation can be caused or contributed by the salmon louse, Lepeophtheirus salmonis. A Caligus infection or infestation can be caused or contributed to by the sea lice, Caligus clemensi, Caligus elongatus, and Caligus rogercresseyi . In various aspects, the fish feed can be for species belonging to the family Salmonidae. For example, the fish feeds herein can be a Salmo and/or Oncorhynchus spp. fish feed. However, it will be appreciated that the fish feeds herein can be given to any fish that is susceptible to a Lepeophtheirus or Caligus infection or infestation.

Pest Control Agents

[0080] The pest control agents suitable for use in the fish feeds and pest control agent compositions herein can adversely affect pests that feed off their hosts. It will be appreciated that when a host fish has consumed the pest control agents as a component of their daily diet for a given duration, it can be transferred to the body of the pest when that pest takes a meal from the host. The pest control agents can include any functional agent or active agent that affects, facilitates, or contributes to the eradication or reduction of a pest infection or pest infestation of a fish or population of fish. Additionally, suitable pest control agents can alleviate or improve one or more of the symptoms associated with a pest infection or pest infestation, as a result of reducing, preventing, or controlling an infection or infestation. Pest control agents for use herein can be biologically active to one or more fish pests and for one or more fish species.

[0081] It will be appreciated that when the fish consume the pest control agents described herein, the pest control agent is systemically distributed throughout the tissues and fluids of the fish. Pests can be exposed to the pest control agents upon ingestion of the pest control agent through the skin, flesh, blood, mucus, mucous membranes, or other tissues of the host organism. Modulation of the pests, such as modulation of the pest behavior and life cycle occur to reduce, prevent, or control the pest infection or infestation in the fish. In various aspects, the pests can be repelled or killed by the pest control agents herein. Thus, the pest control agents herein can be provided to the pests in a fish feed or a pest control agent composition in an amount sufficient to modulate the behavior of the pests.

[0082] Modulation of the pests can have many effects on the pest population, including an ultimate reduction in the number of viable pests available to infect or infest the host fish. Modulation of the pests can include a modulation of the mortality of the pests. It will be appreciated that modulation of the mortality in the pests can include a decrease in the number of viable pests present on the fish or in the fish habitat. Modulation of the pests can further include modulation of pest behavior, including a change in feeding habits, a change in feeding patterns, a change in appetite, a change in mobility patterns, a change in swimming and migration patterns, a change in mating patterns, a change in development, a change in fertility, or any combination thereof, as compared to pests found on control fish not fed a pest control agent. The change in feeding patterns can include a decrease in feeding patterns. The change in appetite can include a decrease in appetite. The change in mobility can include a decrease in mobility. The change in swimming and migration patterns can include a decrease in swimming and migration due to lethargy and lack of energy. The change in mating patterns can include a decrease in mating patterns, which in turn can lead to a decrease in development or production of offspring. The change in development can include an inhibition of development due to an inhibition of the molting process leading to a decrease in development in the pests or a delay in development in the pests. The change in fertility can include an inhibition of or delay in egg production, an inability to produce viable eggs, or a reduction in the total number of gravid female pests.

[0083] In various aspects, modulation of the pests can include a change in development of the pests through their life cycle, including modulation of growth or progression through a particular life stage, modulation of growth or progression from one life stage to the next life stage (e.g., modulating molting), modulation of egg production, modulation of fertility, or any combination thereof. Modulation of growth or progression through a particular life stage can include halting the growth of the organism and preventing further physical development including a decrease in size or sexual development. In some aspects, modulation of growth or progression from one life stage to the next life stage can include preventing the pests from transitioning from one life stage to the next by inhibiting the molting process. Modulation of egg production can include decreasing the production of eggs by females, which can further result in a decrease in fertility of the adult females. Modulation of fertility can include decreasing the fertility of both female and male pests.

[0084] In various aspects, the administration of pest control agents to fish as described herein further can have a beneficial effect on the fish. The administration of the pest control agents can impart a beneficial effect by improving fish welfare by reducing the parasitic load, or total number of parasites, in a given environment around the fish. The administration of the pest control agents can impart a beneficial effect by a reduction in the overall mortality within a fish population by lessening or reducing the impact of a parasitic infection or infestation on the fish population. The administration of the pest control agents can impart a beneficial effect by minimizing or altogether eliminating the impact on the quality and quantity of fish flesh within the fish population. [0085] The fish feeds described herein can include, or be supplemented with, one or more pest control agents. Where a fish feed includes at least two or more different pest control agents, each pest control agent can be individually active (or biologically active) and capable of modulating one or more of the behavior, development, or fertility of a pest. Alternatively, the pest control agents can be a component of a pest control agent composition that can be fed separately to fish. Each pest control agent can be individually effective against one or more different pests as described herein.

[0086] Pest control agents suitable for use in the fish feeds and pest control agent compositions herein can include one or more active agents, including synthetic or natural agents. The one or more synthetic or natural agents can include agents classified as an active pharmaceutical ingredient, a veterinary medicinal product, and the like. In some aspects, the active agent for the pest control agents herein can be obtained from a plant belonging to the genus Azadirachta. The pest control agent can be obtained or extracted from Azadirachta indica - a tree commonly known as the “Neem” tree. Extracts prepared from plants belonging to the genus Azadirachta (e.g., Azadirachta indica) can include potent terpenoid compounds, including one or more azadirachtinoids. The azadirachtinoids include azadirachtin compounds such as azadirachtin A, azadirachtin B, azadirachtin D, azadirachtin E, azadirachtin F, azadirachtin G, azadirachtin H, azadirachtin I, azadirachtin K, and/or other azadirachtin variants. The extracts from plants belonging to the genus Azadirachta can also include many other components in various quantities. In some aspects, the extracts can include additional compounds such as the limonoids salannin, nimbin, deacetyl salinin, and 6-desacetylnimbin. In various aspects, the extracts can further include one or more azadirachtinins.

[0087] As used herein, the term “azadirachtin” can refer to the collective term applied to a large group of active compounds and is intended to encompass not only all naturally occurring variants or derivatives of azadirachtin, including but not limited to azadirachtins A, B, D, E, F, G, H, I, K, but also all synthetic variants, fragments, analogues, and derivatives thereof. In this regard, it will be appreciated that any azadirachtin variants, fragments, derivatives, or analogues for use herein should be functional, in that they exhibit at least one inhibitory effect as described.

[0088] Azadirachtin can be obtained or extracted from any part of Azadirachta indica plant including, for example, the leaves, stems, bark, fruit, seeds, or any combinations thereof by one or more extraction processes. Suitable methods of extraction can include techniques that exploit mechanical pressing of neem seeds (i.e., kernels) and the use of non-polar solvents. Various solvent extraction techniques exploiting alcohol or an aqueous extraction process, mechanical pressing, and non-polar extraction methods can be used to produce azadirachtin A-rich pest control agents for use herein and are described in U.S. Pat. No. 4,556,562; U.S. Pat. No. 5,695,763; and U.S. Pat. No. 11,096,404; the contents of which are incorporated herein by reference in their entirety.

[0089] For example, azadirachtin can be effectively recovered from the seeds of the Neem tree. An exemplary method to recover azadirachtin from neem seeds can include providing neem seeds, crushing the neem seeds, extracting azadirachtin from the crushed seeds with water, and then extracting azadirachtin from the water by adding a second extraction solvent including a nonaqueous solvent that is not miscible with water and has a higher solubility of azadirachtin than water or a surfactant having a turbidity temperature between 20 °C and 80 °C. The concentrated azadirachtin can be recovered from the second extraction solution and shows high activity as an insecticide and parasiticide. Extraction methods employing polar solvents (e.g. water) lead to extracts that are rich in polar components, such as azadirachtin compounds.

[0090] In various aspects, the azadirachtin suitable for use herein includes azadirachtin A, which is by its scientific name of dimethyl [2a7?- [2aa,3B,4B(la/?*,25*,3a *,6a5*,75*,7a *),4aB,5a, 7aS*,8B(E),10B,10aa,10bB]]-10-

(acetyloxy)octahydro-3,5-dihydroxy-4-methyl-8-[(2-methyl- l-oxo-2-butenyl)oxy]-4- (3a,6a,7,7a)-tetrahydro-6a-hydroxy-7a-methyl-2,7-methanofuro [2,3-Z>]oxireno[e]oxepin-la(2Z/)- yl)- IT/,77/-naphtho-[ l ,8-/ic:4,4a-c ‘]difuran-5,10a(8J7)-dicarboxylate.

[0091] Azadirachtin A is the most abundant of a group of the azadirachtinoids. Azadirachtin A makes up about 80% of the azadirachtinoids in the neem seed kernel. The structural formula of azadirachtin A is: [0092] The pest control agents herein can include neem extracts that are an aqueous extract. In various aspects, the neem extract can include an aqueous extract of neem seed. The aqueous extract of neem seed can include an aqueous extract of the neem seed kernel. The aqueous extract of neem seed can include an aqueous extract of the entire neem seed, including the neem seed kernel and the neem seed coating. The aqueous extract of neem seed can be in liquid form, or it can be dried to remove water to create a powder form. By way of example, the neem extracts herein can include an aqueous extract of neem seed or an aqueous extract of neem seed kernel that has been dried into a powder.

[0093] It will be appreciated that the pest control agents described herein are not the same thing as neem extracts described as neem oil or solvent-first neem extracts. In various aspects, the pest control agents herein including azadirachtin are richer in the azadirachtinoid active ingredients, and in particular azadirachtin A, than are neem oil and other oil-based formulations. This is due to the fact that azadirachtinoids, such as azadirachtin A, are relatively polar complex terpenoids with a large number of oxygen functionalities, which make the molecules moderately water-soluble (e.g., a solubility of approximately 2 g/L). As a result, azadirachtinoids such as azadirachtin A are present in much higher concentrations in the extracts obtained employing polar solvents than in neem oil or solvent-first neem extracts. Without wishing to be bound by any particular theory, it is believed that the bioavailability of the active ingredients to the target parasite in the water-based extract of azadirachtin A rich extracts of the present disclosure can be greater than in neem oil given the increased solubility and miscibility of the water-based extract in water. Thus, the pest control agents herein do not, comprise, consist, or consist essentially of, neem oil. The pest control agent of the fish feed provided herein can comprise, consist, or consist essentially of azadirachtin A.

[0094] The pest control agents including neem extract rich in azadirachtin A can include those having from at least 15 wt. % to 33 wt. % azadirachtin A. In various aspects, pest control agents including neem extract rich in azadirachtin A can include those having from at least 20 wt. % to 26 wt. % azadirachtin A. In various aspects, pest control agents including neem extract rich in azadirachtin A can include those having from at least 28 wt. % to 31 wt. % azadirachtin A. In some aspects, pest control agents including neem extract rich in azadirachtin A can include those having from at least 29 wt. % to 30 wt. % azadirachtin A. In other aspects, pest control agents including neem extract rich in azadirachtin A can include those having from at least 34 wt. % to 40 wt. % azadirachtin A. In various aspects, pest control agents rich in azadirachtin A can include those having from 30 ± 1 wt. % azadirachtin A. In various aspects, pest control agents rich in azadirachtin A can include those having from 15 wt. %, 16 wt. %, 17 wt. %, 18 wt. %, 19 wt. %, 20 wt. %, 21 wt. %, 22 wt. %, 23 wt. %, 24 wt. %, 25 wt. %, 26 wt. %, 27 wt. %, 28 wt. %, 29 wt. %, 30 wt. %, 31 wt. %, 32 wt. %, or 33 wt. %, or any amount falling within a range of any of the forgoing. In yet other aspects, pest control agents including neem extract rich in azadirachtin A can include those having from at least 34 wt. % to 45 wt. % azadirachtin A, or at least 38 wt. % to 43 wt. %. As used herein, the terms “neem extract rich in azadirachtin A” and “azadirachtin A- rich composition” can be used interchangeably unless otherwise noted. A composition of an exemplary neem extract rich in azadirachtin A pest control agent suitable for use herein can include the formula as outlined in Table 1.

Table 1. Exemplary Azadirachtin-A Rich Pest Control Agent Formulation

[0095] The pest control agent including a neem extract rich in azadirachtin A can further include other azadirachtinoids at various concentrations. The azadirachtinoids can include azadirachtin compounds such as azadirachtin B at from < 19.0 % w/w, or from < 6.0 % w/w, or from 4.0 to 6.0 % w/w, or from 5.6 % w/w to 6.0 % vil'W, azadirachtin D at from < 13.0 % w/w, or from < 5.0 % w/w, or from 2.5 to 5.0, or from 4.0 % w/w to 5.0 % vil'W, azadirachtin E at from < 5.0 % w/w, or from 1.0 % w/w to 5.0 % w/w, or from 1.5 % w/w to 2.0 % w/w; azadirachtin F at from < 5.0 % w/w, or from 1.0 % w/w to 5.0 % w/w, or from 1.5 % w/w to 2.0 % w/w; azadirachtin G at from < 5.0 % w/w, or from 1.0 % w/w to 5.0 % w/w, or from 1.5 % w/w to 2.0 % w/w; azadirachtin H at from < 5.0 % w/w, or from 1.0 % w/w to 5.0 % w/w, or from 2.5 % w/w to 4.0 % w/w; azadirachtin I at from < 5.0 % w/w, or from 1.0 % w/w to 4.0 % w/w, or from 1.5 % w/w to 2.5 % w/w; and azadirachtin K and/or other azadirachtin variants at from < 5.0 % w/w, or from 1.0 % w/w to 5.0 % w/w, or from 2.5 % w/w to 4.0 % w/w. The extracts further can include azadirachtinin at from < 5.0 % w/w, or from 1.0 % w/w to 5.0 % w/w, or from 2.5 % w/w to 4.0 % w/w.

[0096] An exemplary pest control agent suitable for use herein can include an aqueous extract of neem seed that has been dried into a powder. The powder can include the appearance of a fine white powder. The exemplary pest control agent can include azadirachtin A at a concentration of from 17 wt. % to 37 wt. %, azadirachtin B at a concentration of from 0 wt. % to 19 wt. %, and azadirachtin D at a concentration of rom 0 wt. % to 13 wt. %. The exemplary pest control agent further can include trace amounts of other limonoids including nimbin and salannin. [0097] Exemplary pest control agents including azadirachtin A rich compositions include, but are not to be limited to, NeemAzal® (Coromandel, Inti. Ltd., Telangana, India) or NeemAzal® Technical (Coromandel, Inti. Ltd., Telangana, India), or any derivatives of combinations thereof.

Fish Feeds Containing Azadirachtin A

[0098] The fish feeds herein can include those that are supplemented with the pest control agent azadirachtin A. The fish feeds can be administered to various fish as part of a fish feed diet to control arthropod pests within an aquaculture environment. The fish feeds can be at least partially coated on an exterior surface with an azadirachtin A-rich composition or completely coated on an exterior surface with an azadirachtin A-rich composition. In some aspects, the fish feeds herein can include an azadirachtin A-rich composition that is at least partially dispersed throughout the fish feed. In various aspects, a solid feed such as a base feed pellet, can further include an azadirachtin A-rich composition disposed on the surface or distributed throughout the fish feed, such as within an oil disposed within a porous matrix of on an exterior surface of the base feed pellet. In various aspects, the fish feeds herein can be at least partially coated on an exterior surface with an azadirachtin A-rich composition and further can have an azadirachtin A- rich composition at least partially dispersed throughout the fish feed. In various aspects, the fish feed can include one or more layers of azadirachtin A-rich composition on an exterior surface.

[0099] The fish feeds herein can include an azadirachtin A-rich composition at a concentration from about 0.01 - 100 grams per kilogram (g/kg) fish feed, about 90 g/kg fish feed, about 80 g/kg fish feed, about 70 g/kg fish feed, about 60 g/kg fish feed, about 50 g/kg fish feed, about 40 g/kg fish feed, about 30 g/kg fish feed, about 20 g/kg fish feed, about 0.01-10 g/kg fish feed, about 1- 10 g/kg fish feed, about 2-9 g/kg fish feed, about 3-7 g/kg fish feed, about 4-6 g/kg fish feed, or about 5 g/kg fish feed.

[0100] In various aspects, the fish feeds herein can include an azadirachtin A-rich composition at a concentration from about 0.01 g azadirachtin A-rich composition per kilogram fish feed (g/kg), 0.05 g/kg fish feed, 0.1 g/kg fish feed, 0.2 g/kg fish feed, 0.3 g/kg fish feed, 0.4 g/kg fish feed, 0.5 g/kg fish feed, 0.6 g/kg fish feed, 0.7 g/kg fish feed, 0.8 g/kg fish feed, 0.9 g/kg fish feed, 1.0 g/kg fish feed. 1.25 g/kg fish feed, 1.5 g/kg fish feed, 1.75 g/kg fish feed, 2.0 g/kg fish feed, 2.25 g/kg fish feed, 2.5 g/kg fish feed, 2.75, g/kg fish feed, 3.0 g/kg fish feed, 5.0 g/kg fish feed. 5.25 g/kg fish feed, 5.5 g/kg fish feed, 5.75 g/kg fish feed, 6.0 g/kg fish feed, 6.25 g/kg fish feed, 6.5 g/kg fish feed, 6.75, g/kg fish feed, 7.0 g/kg fish feed, 7.0 g/kg fish feed. 7.25 g/kg fish feed, 7.5 g/kg fish feed, 7.75 g/kg fish feed, 8.0 g/kg fish feed, 8.25 g/kg fish feed, 8.5 g/kg fish feed, 8.75, g/kg fish feed, 9.0 g/kg fish feed, 9.25 g/kg fish feed, 9.5 g/kg fish feed, 9.75, g/kg fish feed, 10.0 g/kg fish feed, 15 g/kg fish feed, 20 g/kg fish feed, 25 g/kg fish feed, 30 g/kg fish feed, 35 g/kg fish feed, 40 g/kg fish feed, 45 g/kg fish feed, 50 g/kg fish feed, 55 g/kg fish feed, 60 g/kg fish feed, 65 g/kg fish feed, 70 g/kg fish feed, 75 g/kg fish feed, 80 g/kg fish feed, 85 g/kg fish feed, 90 g/kg fish feed, 95 g/kg fish feed, 100 g/kg fish feed, or any amount within a range of any of the forgoing concentrations.

[0101] It will be appreciated that the aforementioned azadirachtin A-rich composition concentrations equate to about from 0.001-10 (weight percent) % w/w azadirachtin A-rich composition to fish feed. In various aspects, the fish feeds herein can include an azadirachtin A- rich composition at from 0.001 % w/w, 0.002 % w/w, 0.003 % w/w, 0.004 % w/w, 0.005 % w/w, 0.006 % w/w, 0.007 %w/w, 0.008 % w/w, 0.009 % w/w, 0.010 % w/w, 0.020 % w/w, 0.030 % w/w, 0.040 % w/w, 0.050 % w/w, 0.060 % w/w, 0.070 % w/w, 0.080 % w/w, 0.090 % w/w, 0.10 % w/w, 0.11 % w/w, 0.12 % w/w, 0.13 % w/w, 0.14 % w/w, 0.15 % w/w, 0.16 % w/w, 0.17 % w/w, 0.18 % w/w, 0.19 % w/w, 0.20 % w/w, 0.21 % w/w, 0.22 % w/w, 0.23 % w/w, 0.24 % w/w, 0.25 % w/w, 0.26 % w/w, 0.27 % w/w, 0.28 % w/w, 0.29 % w/w, 0.30 % w/w, 0.31 % w/w, 0.32 % w/w, 0.33 % w/w, 0.34 % w/w, 0.35 % w/w, 0.36 % w/w, 0.37 % w/w, 0.38 % w/w, 0.39 % w/w, 0.40 % w/w, 0.41 % w/w, 0.42 % w/w, 0.43 % w/w, 0.44 % w/w, 0.45 % w/w, 0.46 % w/w, 0.47 % w/w, 0.48 % w/w, 0.49 % w/w, 0.50 % w/w, 0.51 % w/w, 0.52 % w/w, 0.53 % w/w, 0.54 % w/w, 0.55 % w/w, 0.56 % w/w, 0.57 % w/w, 0.58 % w/w, 0.59 % w/w, 0.60 % w/w, 0.61 % w/w, 0.62 % w/w, 0.63 % w/w, 0.64 % w/w, 0.65 % w/w, 0.66 % w/w, 0.67 % w/w, 0.68 % w/w, 0.69 % w/w, 0.70 % w/w, 0.71 % w/w, 0.72 % w/w, 0.73 % w/w, 0.74 % w/w, 0.75 % w/w, 0.76 % w/w, 0.77 % w/w, 0.78 % w/w, 0.79 % w/w, 0.80 % w/w, 0.81 % w/w, 0.82 % w/w, 0.83 % w/w, 0.84 % w/w, 0.85 % w/w, 0.86 % w/w, 0.87 % w/w, 0.88 % w/w, 0.89 % w/w, 0.90 % w/w, 0.91 % w/w, 0.92 % w/w, 0.93 % w/w, 0.94 % w/w, 0.95 % w/w, 0.96 % w/w, 0.97 % w/w, 0.98 % w/w, 0.99 % w/w, 1.0 % w/w, 2.0 % w/w, 3.0 % w/w, 4.0 % w/w, 5.0 % w/w, 6.0 % w/w, 7.0 % w/w, 8.0 % w/w, 9.0 % w/w, or 10.0 % w/w (i.e., weight azadirachtin A-rich composition to weight fish feed), or any amount within a range of any of the forgoing values.

[0102] In various aspects herein, the fish feeds can be administered to the fish having an azadirachtin A-rich composition at a concentration selected from the group including 0.05 % w/w,

0.06 % w/w, 0.07 % w/w, 0.08 % w/w, 0.09 % w/w, 0.10 % w/w, 0.11 % w/w, 0.12 % w/w, 0.13 % w/w, 0.14 % w/w, 0.15 % w/w, 0.16 % w/w, 0.17 % w/w, 0.18 % w/w, 0.19 % w/w, 0.20 % w/w,

0.30 % w/w, 0.40 % w/w, 0.50 % w/w, 0.60 % w/w, 0.70 % w/w, 0.80 % w/w, 0.90 % w/w, or 1.0 % w/w (i.e., weight azadirachtin A-rich composition to weight fish feed), or a range within any of the forgoing concentrations.

[0103] Azadirachtins can be relatively unstable in water, however when they are a component of a fish feed, such as dispersed throughout or coated thereon, the azadirachtins, including azadirachtin A, are rendered at least temporarily stable such that they can exhibit their full biological activity during feeding. Any fish feed that falls to the ocean floor will degrade upon prolonged exposure to water. In addition, it should be noted that azadirachtin A, or any of the neem extract agents of the present disclosure exhibit minimal risk of toxic effects on fish or humans and are therefore safe to use in both wild fish and farmed fish stocks. While arthropods and other invertebrates are sensitive to the active ingredient (i.e. azadirachtin A), higher organisms, including mammals, are unaffected. Furthermore, since azadirachtin A or any neem extract as described herein are readily soluble in water, they do not reside and accumulate in fish. Rather, once administration has ceased, the pest control agent can quickly lose effectiveness, as it is metabolized, degraded, and/or excreted. In the case of farmed fish stocks, this ensures a little to no withdrawal period to harvest following administration of a fish feed or composition as described herein.

[0104] The fish feed provided herein can include an azadirachtin A-rich composition together with one or more other agents. The one or more or other agents can include anti-ectoparasitic agents, antimicrobial agents (e.g., antibiotic, antibacterial, antifungal, antiviral agents), antiparasitic agents (e.g., anti-endoparasitic agents or anti-ectoparasitic agents), or antiprotozoal agents. The one or more other agents can be mixed with or coated on, or layered within, the fish feed. The one or more other agents can be provided separately (e.g., either in liquid or solid form) and can be administered separately (e.g., before or after) or concurrently with (e.g., during) a fish feed.

Method of Making a Fish Feed

[0105] The disclosure herein provides a method of making a fish feed including one or more pest control agents, such as neem extracts including azadirachtin A-rich compositions. The method can include the step of providing a base feed and applying a quantity of pest control agent to a surface of the base feed. A base feed can be formed from various raw materials as described elsewhere herein. For example, the method can include coating the base feed with a quantity of pest control agent. The base feeds can be at least partially coated on an exterior surface with a pest control agent or completely coated on an exterior surface with a pest control agent. In some aspects, the base feeds herein can include a pest control agent that is at least partially dispersed throughout the base feed. In various aspects, the base feeds herein can be at least partially coated with a pest control agent and further can have a pest control agent at least partially dispersed throughout the base feed. In some aspects, the pest control agent can be distributed throughout the base feed, such as throughout a porous matrix of the base feed. In various aspects, the base feed can be coated on an exterior surface with more than one layer of pest control agent, where each layer can include the same pest control agent, or in some aspects each layer can include a different pest control agent.

[0106] The pest control agent can be incorporated into or mixed into the base feed by various processes. In various aspects, the base feed can be made using an extrusion process or a pressing process. The pest control agent can be mixed with the base during its manufacture such that it becomes distributed through all or a part of the fish feed. Once the pest control agent has been mixed with the base feed, the base feed and pest control agent mixture can be formed into, for example, pellets, flakes, tablets, powders, or any form as desired. In the case of temperature sensitive pest control agents, such pest control agents can be added to a base feed after it has been formed into one of the various forms as indicated. In various aspects, the pest control agent can be sprayed onto a base feed that has already been formed into pellets, flakes, tablets, and the like. For example, the pest control agent can be applied to a base feed as one or more layers or top coatings. In various aspects, the pest control agent can be applied to an outside surface of a pellet or a flake - in this way a fish feed pellet or flake can become wholly or partially coated with the pest control agent. One or more layers or coatings of agent can be applied to an outside surface of a fish feed flake or pellets. Any layer or coating of agent can be “sealed” or protected by the application of one or more additional coatings or layers of a sealing substance. In various aspects, the pest control agent herein can be dispersed in one or more oils or fractions thereof and can be incorporated into a porous matrix within the fish feed by a vacuum coating process.

[0107] By way of example, a layer or coating of agent can be sealed by the application of a layer or coating of oil, such as fish oil. In various aspects, one or more further layers or coatings of fish feed can be applied to the (optionally sealed) coating or layer of fish feed. In this way, any given fish feed flake or pellet can include multiple layers of fish feed, sealing substance and/or pest control agent layers. It will be appreciated that the fish feeds described herein can be at least partially coated on an exterior surface with pest control agent. In various aspects, the fish feeds herein can include a pest control agent that is at least partially dispersed throughout the fish feed. In various aspects, the fish feeds herein can include a pest control agent that is at least partially coated with pest control agent and at least partially dispersed throughout the fish feed.

[0108] The method for incorporating the pest control agent into the fish feed can include incorporating the pest control agent, such that the final concentration of pest control agent in the fish feed includes from about 0.01 gram pest control agent per kilogram of fish feed (g/kg) to about 1000 g/kg, or from about 0.01 g/kg, 0.1 g/kg, 1 g/kg, 2 g/kg, 3 g/kg, 4 g/kg, 5 g/kg, 6 g/kg, 7 g/kg, 8 g/kg, 9 g/kg or 10 g/kg, 20g/kg, 30g/kg, 40g/kg, 50 g/kg, 60 g/kg, 70 g/kg, 80 g/kg, 90 g/kg, 100 g/kg, 250 g/kg, 500 g/kg, 750 g/kg, or 1000 g/kg, or any amount within a range of any of the forgoing. Concentrations are described herein in more detail in reference to the fish feed.

[0109] The method for incorporating the pest control agent into the fish feed can include determining the final concentration of pest control agent that is incorporated as a part of the fish feed. The determination of the final concentration can include sampling the fish feed using various quantitative analytical methods. By way of example, the fish feed samples can be extracted by a process of overnight protein precipitation in methanol. Following extraction the sample can be cleaned with Supel™ QuE Z-Sep+ (Sigma Aldrich, St. Louis, Missouri, USA) sorbent, which is a silica gel-based material having active zirconia-based phase, a particle size of approximately 50 pm, and a 70-angstrom (A) pore size. The resulting extraction solution can be filtered through polytetrafluoroethylene filters having a pore size from 0.2 pm or greater. Analysis of the final concentration of pest control agent in the fish feed samples can be performed using high performance liquid chromatography with ultraviolet detection (HPLC-UV).

[0110] The methods herein can include extracting a neem extract rich in azadirachtin A, including those having from at least from 15 wt. % to 33 wt. % azadirachtin A, from at least 28 wt. % to 31 wt. % azadirachtin A, and from at least 29 wt. % to 30 wt. % azadirachtin A.

[0111] The method further can include the step of sealing the azadirachtin A-rich composition applied to a surface of the fish feed. The azadirachtin A-rich composition can be sealed by applying a coating of fish oil to the azadirachtin A-rich composition coated fish feed. Any sealing substance used to seal the azadirachtin A-rich composition can be applied such that it coats all or a part of the azadirachtin A-rich composition coating.

Pest Control Agent Compositions

[0112] The present disclosure further provides a pest control agent composition for administration to fish, where the pest control agent composition can include one or more pest control agents. It will be appreciated that the pest control agent compositions are not a fish feed and are intended for separate or supplemental administration to fish in addition to a fish feed. The pest control agent compositions can be provided separately for administration before, during, or after administration of the fish feeds. Accordingly, in various aspects, the pest control agent compositions herein can be suitable for use in some aspects as a form of veterinary medicinal product or dietary supplement for reducing, preventing, or controlling pest infections or pest infestations in fish. The pest control agent compositions can be administered to the fish at the concentrations described elsewhere herein. For example, the pest control agent compositions herein can be administered at from about 0.01 grams pest control agent per kilogram of fish feed (g/kg) to about 100 g/kg, as described elsewhere herein. In various aspects, the pest control agent composition includes a neem extract rich in azadirachtin A.

[0113] The pest control agent compositions herein can include azadirachtin extracts rich in azadirachtin A. The pest control agent composition can include a liquid, solid, or semi-solid form, and further can include one or more of an excipient, diluent, carrier, vitamins, minerals, or combinations thereof. The pest control agent compositions can be in the form of a dietary supplement that is provided as any of granules, flakes, pellets, powders, tablets, pills, capsules, and the like. In various aspects, the pest control agent compositions herein can be formed into many shapes and sizes. In various aspects, the fish feeds herein can be in the shape of a triangle, a square, a rectangle, a sphere, a diamond, a cylinder, a pellet, a clover, an amorphous shape, and the like. The fish feeds can be formed by a process including one or more of extrusion, retort, cold-pressing, high-pressure processing, and the like.

[0114] Alternatively, the pest control agent composition can be provided in a form that is edible by fish but that does not provide nutrition to the fish. By way of example, the pest control agent composition can include a veterinary medicinal product that can include substances or combinations of substances to manage or prevent diseases in fish. The pest control agent composition can also be formulated for parenteral administration. Thus, the pest control agent composition can include pharmaceutically acceptable carriers, diluents, or excipients, or combinations thereof. Furthermore, the pest control agent composition can be sterile.

[0115] The pest control agent compositions herein can be included in one or more types of fish feed designed for mixing with another composition, such as a base feed. The pest control agent composition can be in the form of a premix, a concentrate, a base mix, a supplement, a top dress, liquid drench, or a combination thereof.

[0116] The pest control agent in the pest control agent compositions herein can include one or more agents for reducing, preventing, or controlling an infection or infestation caused or contributed by one or more endoparasite or ectoparasite pests, including any type of worms, helminths, flukes, lice, mites, bacteria, viruses, fungi, and protozoa, as described elsewhere. Each pest control agent included in the pest control agent compositions can be individually capable of reducing, preventing, or controlling one or more of a parasitic, bacterial, viral, fungal, or protozoal infections or infestations. By way of example, the pest control agent compositions herein can include those exhibiting one or more inhibitory effects, including an antiparasitic effect, an antibacterial effect, an antiviral effect, an antifungal effect, an antiprotozoal effect, or any combinations thereof.

[0117] A pest control agent composition can be administered before during or after the administration of any of the fish feeds. In some aspects, the pest control agent compositions can be administered with fish feed that does not contain a pest control agent. In some aspects, the pest control agent compositions can be administered in conjunction with fish feed that does contain a pest control agent. When used in conjunction with fish feed that does contain a pest control agent, the separate pest control agent composition can include the same pest control agent as in the fish feed or it can be a different pest control agent than in the fish feed. When used in conjunction with fish feed that does contain a pest control agent, the separate pest control agent composition can be the same concentration as the pest control agent in the fish feed or it can be a different concentration than the pest control agent in the fish feed. The pest control agent compositions herein can be included in the diet of fish in the form of a veterinary medicinal product or dietary supplement to any complete and balanced fish feed or can be provided as a component of a complete fish feed.

Methods of Administration of Pest Control Agents to Fish

[0118] The pest control agents herein can be administered to fish in the fish feeds and pest control agent compositions. Management methods that utilize the pest control agents within fish feeds can be referred to as in-feed agent delivery methods. Thus, the present disclosure provides in-feed agent delivery methods for reducing, preventing, or controlling pests. It will be appreciated that an in-feed agent delivery method is not a process that applies the pest control agents topically to the target pests. In-feed agent delivery methods herein can further utilize pest control agents in a non-feed form such as a veterinary medicinal product or dietary supplement. Thus, the present disclosure further provides veterinary medicinal products or dietary supplements as agents for reducing, preventing, or controlling pests. [0119] The pest control agents that are not included in fish feed can be administered to the fish in a separate pest control agent composition as a complement to fish feed, such as in the form of a veterinary medicinal product or a dietary supplement. The fish feed can be administered at the same time or separately from a pest control agent composition. It should be noted that the various pest control agents herein can be administered to fish that are sick, fish that are infested with parasites, fish that are otherwise healthy in order to prevent parasitic infection, or fish that are less aggressive due to a different infection or condition not associated with a pest infection or infestation. It will be understood that fish that are sick or less aggressive may eat less and therefore may consume lower concentrations of the pest control agent. Thus, management methods that are based on the use of both fish feeds and pest control agent compositions can be particularly useful for managing fish whose appetites are affected by illness, infection, infestation, or being a less aggressive fish that generally eats less fish feed. Moreover, in less aggressive, or low feeding fish, the concurrent use of a pest control agent composition with fish feed supplemented with a pest control agent can boost or ensure the correct pest control agent concentration is administered to fish.

[0120] The concentration of pest control agent added to fish feeds or pest control agent compositions herein can be an amount effective to achieve the desired modulation of the behavior, development, or mortality of the pests as discussed elsewhere herein. It will be appreciated that the exact amount of pest control agent to be added to a fish feed or pest control agent compositions herein can vary depending on, for example, the species of fish, the number of fish to be fed, the extent of the infection or infestation, and the like. Other factors that influence the amount of pest control agent added to the fish feeds or pest control agent compositions include, for example, the presence of possible competitors for the feed (i.e. other non-target animal species that can eat the fish feed), the type of pest to be controlled, the age and maturity of the pests, the age and maturity of the fish, the season, the water type (e.g., pH, salinity, purity, temperature), and the aggressiveness of the fish. It will be appreciated that the concentration of pest control agents added to a fish feed or pest control agent composition herein can include an amount effective to achieve a desired effect to modulate the behavior and development of the pests, where the amount effective includes one or more concentrations or ranges of concentrations as recited herein. It will be appreciated that the effective amount effective can be determined by performing a comparison to a control fish or group of fish not fed the pest control agents.

[0121] The fish feeds and pest control agent compositions can be formulated such that the concentration of the pest control agent administered to the fish through the fish feed or pest control agent compositions can be approximately 0.01-100 mg pest control agent per kg body weight/day (mg/kg/day), 1-90 mg/kg/day, 1-80 mg/kg/day, 1-70 mg/kg/day, 1-60 mg/kg/day, 5-50 mg/kg/day, 10-40 mg/kg/day, 15-35 mg/kg/day, 20-30 mg/kg/day, 0.01-10 mg/kg/day, or 0.01-5.0 mg/kg/day. In various aspects, the pest control agent is administered to the fish in the fish feed at a targeted concentration from 0.01 mg to 5.0 mg azadirachtin A per kg body weight per day. In various aspects, the pest control agent is administered to the fish in the fish feed at a targeted concentration from 1.5 mg to 2.5 mg azadirachtin A per kg body weight per day. In some aspects, the pest control agent is administered to the fish in the fish feed at a targeted concentration from 2.6 mg to 5.0 mg azadirachtin A per kg body weight per day.

[0122] It will be appreciated that the amount of pest control agent administered to the fish can include an amount effective to produce an inhibitory effect against one or more pests within a range of approximately 0.01-100 mg/kg/day (e.g., mg pest control agent/kg body weight/day) includes at least 0.01 mg/kg/day, 0.02 mg/kg/day, 0.03 mg/kg/day, 0.04 mg/kg/day, 0.05 mg/kg/day, 0.06 mg/kg/day, 0.07 mg/kg/day, 0.08 mg/kg/day, 0.09 mg/kg/day, 0.10 mg/kg/day, 0.20 mg/kg/day, 0.30 mg/kg/day, 0.40 mg/kg/day, 0.50 mg/kg/day, 0.60 mg/kg/day, 0.70 mg/kg/day, 0.80 mg/kg/day, 0.90 mg/kg/day, 1.0 mg/kg/day, 2.0 mg/kg/day, 3.0 mg/kg/day, 4.0 mg/kg/day, 5.0 mg/kg/day, 6.0 mg/kg/day, 7.0 mg/kg/day, 8.0 mg/kg/day, 9.0 mg/kg/day, 10.0 mg/kg/day, 11.0 mg/kg/day, 12.0 mg/kg/day, 13.0 mg/kg/day, 14.0 mg/kg/day, 15.0 mg/kg/day, 16.0 mg/kg/day, 17.0 mg/kg/day, 18.0 mg/kg/day, 19.0 mg/kg/day, 20.0 mg/kg/day, 25.0 mg/kg/day, 30.0 mg/kg/day, 35.0 mg/kg/day, 40.0 mg/kg/day, 45.0 mg/kg/day, 50.0 mg/kg/day, 55.0 mg/kg/day, 60.0 mg/kg/day, 65.0 mg/kg/day, 70.0 mg/kg/day, 75.0 mg/kg/day, 80.0 mg/kg/day, 85.0 mg/kg/day, 90.0 mg/kg/day, 95.0 mg/kg/day, or 100.0 mg/kg/day, or any amount within a range of any of the forgoing values. Any of the aforementioned amounts effective to produce an inhibitory effect can be utilized in the targeted concentrations described herein.

[0123] A fish feed or pest control agent composition can be administered for a period of time for as long as required to achieve the desired inhibitory effect. For example, the pest control agent composition or fish feed can be administered over about a 10 to 20 days, or about 14-18 days. It will be appreciated that, the pest control agent composition, the fish feed, or both, can be administered for consecutive days for 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days, or for any number of days falling within a range of any of the forgoing. In various aspects, the pest control agent composition or fish feeds herein can be administered for a longer period of time, such as past 20 days. In various aspects, the fish feed or pest control agent can be administered for 11 days. In various aspects, the fish feed or pest control agent can be administered for 14 days. It should be understood that the time required for administration of the pest control agent composition or fish feeds herein can be of a variable length in order to target the developmental life stages of the pests present in a fish population, for water temperature, pest control agent concentration, or any combinations thereof. In some aspects, the pest control agents herein could be administered prophylactically in the diet of fish at an amount effective to prevent a pest infection or infestation from taking hold within a population of fish. It will further be appreciated that the pest control agents herein could be administered prophylactically in the diet for any period of time during the fish life cycle, such as from stocking to harvest, seasonally, or during an infection or infestation outbreak within a population or within a nearby farm infection or infestation outbreak.

[0124] In various management methods, the pest control agents herein can be administered for non-consecutive days, where the pest control agent is administered for a predetermined period of time followed by a rest period, and then administered again for a predetermined period of time and followed by a rest period, and so on. By way of example, in some aspects, the pest control agent can be administered for three out of every 10 days. In other aspects, the pest control agent can be administered for seven out of every 14 days. The method for administering the pest control agent for predetermined period of time followed by a rest period can be repeated for as long as desired or until a pest infection or infestation is reduced, prevented, or controlled. It will be appreciated that the pest control agent can be administered for a predetermined period of time, including from 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days out of every 5 days to 30 days of rest in between administration.

[0125] During the period of administration, the pest control agent composition or fish feed can be administered as many times per day as required to achieve the inhibitory effect. For example, the pest control agent composition or fish feeds described herein can be administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times a day.

Targeted Management Methods for Pest Control Agent Administration

[0126] The methods for administering the pest control agents herein can include targeted management methods that utilize a targeted concentration and a targeted duration of exposure to maximize the antiparasitic effects of the pest control agent against various life stages of sea lice. The targeted management methods can include administering a pest control agent through one or more fish feeds or pest control agent compositions as described elsewhere herein. The targeted concentration can include an amount effective to produce an inhibitory effect against one or more pests, where the inhibitory effect can include one or more of an anti-feedancy effect, an antimolting effect, an antifertility and anti-fecundity effect, or an antiparasitic effect. The targeted duration of exposure can include a period of time that is required to achieve the desired inhibitory effect. It will be appreciated that in various aspects, some life stages will require a smaller targeted concentration and targeted duration of exposure, while other life stages will require a larger targeted concentration and longer targeted duration of exposure.

[0127] By targeting the concentration and the duration of exposure at various life stages, the management methods can be tailored to maximize efficacy at the lowest concentration of pest control agent required to elicit an inhibitory effect. By utilizing the lowest concentration of pest control agent possible, the management methods can achieve high efficacy at lower targeted concentrations in order to keep the costs of management low for farmers. As described elsewhere herein, the pest control agents can include those neem extracts rich in azadirachtin A that can produce inhibitory effects against one or more pests including one or more effects for reducing, preventing, or controlling the concentration and spread of various parasites such as sea lice. As described, azadirachtin A has anti-feedant properties against sea lice, where the anti-feedant properties can exert one or more anti-feedancy effects including malnourishment, delayed development, prevention or delay of molting, and death, all of which can contributed to excess mortality of the sea lice at all life stages over time.

[0128] The inhibitory effect can include a reduction in the total number of sea lice available to molt from one developmental life stage to another, such as from the copepodid stage to chalimus 1 stage, the chalimus 1 to chalimus 2 stage, the chalimus 2 to preadult stage, the preadult stage to the adult stage in a sea lice population exposed to fish fed a diet containing azadirachtin A as compared to a population fish infected or infested with one or more pests that are fed a diet lacking the neem extract rich in azadirachtin A. The inhibitory effect can further include a reduction in the total number of adult females or a reduction in total number of adult males in a sea lice population exposed to fish fed a diet containing azadirachtin A as compared to a population fish infected or infested with one or more pests that are fed a diet lacking the neem extract rich in azadirachtin A.

[0129] The inhibitory effect of the pest control agents can lead to excess mortality in the sea lice population. For example, sea lice mortality can increase with each day that the parasites are exposed to the targeted concentrations during the targeted duration of exposure and is referred to herein as “extra daily mortality.” Sea lice mortality also increases at each molting stage and is referred to herein as “extra mortality at molting.” In addition to causing extra daily mortality due to malnourishment, the neem extracts rich in azadirachtin A can substantially inhibit or prevent the development of sea lice from one stage to the next to result in extra mortality at molting. Further, the neem extracts rich in azadirachtin A can exhibit an ovicidal effect on the female pests, where the ovicidal effect can include a decrease in egg production, a decrease in egg viability, a decrease in egg size, and a decrease in time to hatching to further lead to extra mortality at molting. [0130] As pests, including sea lice, molt through the nauplius stage toward the adult stage, they become progressively less susceptible to the neem extract. It has been found that various life stages exhibit a differential response to the targeted concentration, targeted duration of exposure, and timing of pest control agent administration. The methods herein can be configured to target sea lice in the early developmental stages when they are firmly attached to the fish, including the copepodid, chalimus 1, and chalimus 2 life stages. The copepodid, chalimus 1, and chalimus 2 life stages can include lice aged from hatching to, 5-, 10-, 12-, 15-, 17- and 20-day old lice, depending on water temperature and other external environmental conditions. In various aspects, herein, the methods can include administering to the fish a fish feed rich in azadirachtin A at any concentration within a range from 0.01 mg/kg fish/day to 5.0 mg/kg fish/day for 14 days when sea lice are present at the copepodid, chalimus 1, and chalimus 2 life stages. In various aspects, herein, the methods can include administering to the fish a fish feed rich in azadirachtin A at any concentration within a range from 1.5 mg/kg fish/day to 2.5 mg/kg fish/day for 14 days when sea lice are present at the copepodid, chalimus 1, and chalimus 2 life stages. In some aspects, herein, the methods can include administering to the fish a fish feed rich in azadirachtin A at any concentration within a range from 2.6 mg/kg fish/day to 5.0 mg/kg fish/day for 14 days when sea lice are present at the copepodid, chalimus 1, and chalimus 2 life stages. In other aspects, the methods can include administering to the fish a fish feed rich in azadirachtin A at a concentration of about 2 mg/kg fish/day for 14 days when sea lice are present at the copepodid, chalimus 1, and chalimus 2 life stages. In yet other aspects, the methods can include administering to the fish a fish feed rich in azadirachtin A at a concentration of about 1 mg/kg fish/day for 14 days when sea lice are present at the copepodid, chalimus 1, and chalimus 2 life stages.

[0131] The methods herein can be configured to target sea lice present on the fish in the later developmental stages when the sea lice become more mobile and are able to detach themselves from and reattach to the fish at will. These later developmental stages include the preadult 1, preadult 2, and adult life stages, where the lice can be aged from approximately 20 days or more, depending on water temperature and other external conditions. In various aspects, herein, the methods can include administering to the fish a fish feed rich in azadirachtin A at any concentration within a range from 1.0 mg/kg fish/day to 5.0 mg /kg fish/day for 14 days when sea lice are present at the late chalimus 2, preadult 1, preadult 2, and adult life stages. In various aspects, herein, the methods can include administering to the fish a fish feed rich in azadirachtin A at any concentration within a range from 2.5 mg/kg fish/day to 5.0 mg /kg fish/day for 14 days when sea lice are present at the late chalimus 2, preadult 1, preadult 2, and adult life stages. In various aspects, herein, the methods can include administering to the fish a fish feed rich in azadirachtin A at any concentration within a range from 3.5 mg/kg fish/day to 4.5 mg /kg fish/day for 14 days when sea lice are present at the late chalimus 2, preadult 1, preadult 2, and adult life stages. In other aspects, the methods can include administering to the fish a fish feed rich in azadirachtin A at a concentration of about 4 mg/kg fish /day for 14 days when sea lice are present at the late chalimus 2, preadult 1, preadult 2, and adult life stages.

[0132] Targeted management methods of the present disclosure can be tailored to expose the sea lice to pest control agent within one or more development windows to target various or multiple life stages of sea lice. Referring now to FIG. 2, are plots of the exemplary development of sea lice present in a fish population as a function of time and various targeted management models. Plots A-D each represent one targeted management model where a population of fish is infected with sea lice starting in the copepodid life stage at day 0 using the same concentration of pest control agent for each targeted management method. Sea lice mortality (e.g., fraction of sea lice remaining (%)) is presented over a 60-day period following infection at day 0. Plot A represents a no management method control; plot B represents a targeted management method with a pest control agent for 14 days starting at the day of infection (day 0); plot C represents a targeted management method with a pest control agent for 14 days starting at 10 days post infection (day 10); and plot D represents a targeted management method with a pest control agent for 14 days starting at 20 days post infection (day 20). Days of exposure to the pest control agent within a development window are illustrated in plots B-D as dotted vertical lines indicating starting and stopping points. The pest control agent can be administered to the fish through a fish feed or a pest control agent composition as described herein.

[0133] In plot A of FIG. 2, the fish are not administered a pest control agent and thus there is no pest control agent exposure experience by the sea lice. Plot A represents an exemplary progression of sea lice from the copepodid life stage (e.g., day 0) through the adult life stage (e.g., day 25-30). As shown in plot A, the population of sea lice is predominantly (e.g., approximately 100%) present in the copepodid stage at day 0. For the purposes of the plots presented in FIG. 2, all sea lice in the copepodid, chalimus 1, and chalimus 2 life stage are presented as CH in each plot. According to plot A, the sea lice present in the CH life stages gradually transition from CH stages to the preadult 1 life stage (i.e., PA 1 - preadult 1) beginning around day 10. The sea lice continue their progression from PA 1 to preadult 2 (i.e., PA 2 - preadult 2) until the adult stage (i.e., A - adult) is reached by most sea lice in the population by about day 25-30. It will be appreciated that not all sea lice will survive from the CH life stages until adulthood due to natural mortality within the sea lice population.

[0134] In plot B of FIG. 2, the fish are administered a diet containing a pest control agent via a fish feed or a pest control agent composition and the exemplary sea lice population is exposed to pest control agent through starting at day 0 upon infection with copepodids for 14 days. In this particular targeted management method, the sea lice in the CH life stage decrease and fall to zero by about 25-30 days without developing into any appreciable number of sea lice in the preadult or adult life stages. In plot C of FIG. 2, the fish are administered a diet containing a pest control agent via a fish feed or a pest control agent composition and the exemplary sea lice population is exposed to pest control agent through the fish starting at day 10 post infection with copepodids for 14 days. The sea lice in the CH life stages begin to transition from CH life stages to PA 1 life stages by about day 10, however, the PA 1, PA 2, and A life stages do not develop to the same levels when compared to a control population. Sea lice managed according to the targeted management method in plot C experience an increase in excess daily mortality and an excess mortality at molting for various life stages. In plot D of FIG. 2, the fish are administered a diet containing a pest control agent via a fish feed or a pest control agent composition and the exemplary sea lice population is exposed to the pest control agent at 20 days post infection. In this targeted management method including prior to exposure to the pest control agent, sea lice under this have already begun the transition from CH life stages into PA 1 and PA 2 at levels just below those levels for the control population presented in plot A. Notably, the PA 2 sea lice develop under the targeted management method shown in plot D are capable of developing into adults, however, the adult population does not reach the levels found in the control population. The adult population of sea lice in this population exhibits an increase in excess daily mortality between 30 to 60 days after the exposure to the pest control agent is ended, suggesting a prolonged effect on the sea lice post exposure.

[0135] It will be appreciated that the developmental life stage composition of sea lice populations present during an infection or infestation of farmed fish changes in response to various environmental factors, including the season, ocean currents, water temperature, or the use of physical delousing systems. The targeted management methods herein can include alternating between the administration of the pest control agent at the lower range of targeted concentrations and at a higher range of targeted concentrations, depending on the life stage present at a given time. Further, the methods herein can include administering the pest control agents at the onset infection or infestation, part way through the infection or infestation, or later in the infection or infestation, or combinations thereof. It will be appreciated that the onset of the infection or infestation can include from days 0 to 9 post infection with the copepodid life stage; part way through the infection or infestation can include from days 10 to 19 post infection with the copepodid life stage; and later in the infection or infestation can include from days 20 days or more post infection with the copepodid life stage.

[0136] By way of example, some methods can include alternating between administering targeted concentrations from 0.01 mg/kg fish/day to 2.5 mg/kg fish/day for 14 days to target the early life stages including copepodid, chalimus 1, and chalimus 2 life stages, followed by administering targeted concentrations from 2.6 mg/kg fish/day to 5.0 mg /kg fish/day for 14 days to also target the later life stages including preadult 1, preadult 2, and adult life stages. It will be appreciated that the infection or infestation can be targeted to remove the attached copepodid, chalimus 1, and chalimus 2 life stages of sea lice during a first exposure at a first pest control agent concentration followed by a second exposure at a second pest control agent concentration targeted to also remove the later life stages of mobile sea lice. It will further be appreciated that a targeted concentration tailored to target later life stages can be sufficient to also target early life stages, however, utilizing a high concentration for the early life stages can be unnecessary and more costly to fish farmers.

[0137] The targeted management methods tailored to target sea lice in the early life stages and later life stages can be used as a component of a comprehensive sea lice management regime that further includes the use of one or more physical delousing systems or any other mechanism that selectively kills mobile preadult and adult life stages. A number of physical delousing systems have been developed and are utilized to remove pests from fish infected or infested with one or more pests as another strategy to utilize in conjunction with various chemotherapeutic agents. The physical delousing systems can include one or more of an aqueous management system, a lightbased management system, a thermal management system, a mechanical management system, or a cleaner fish-based management system. In various aspects, the targeted management methods herein include one or more methods that include coordinating the timing of the administration of pest control agents with one or more physical delousing systems. In some aspects, the methods herein include one or more phases that include the administration of pest control agents with one or more physical delousing systems and to reduce the infection pressure within a given geographical region.

[0138] In various aspects, the use of the pest control agents herein can prolong the time between physical delousing exposures and can further reduce the total number of physical delousing exposures required during a production cycle of a fish from stocking of the fish in the sea cages to harvest time. In various aspects, each targeted management methods using the pest control agent compositions herein can reduce the number of physical delousing exposures experienced by the fish overall by from 1 to 5 exposures. In various aspects, each targeted management method exposing the fish to the pest control agent compositions herein can reduce the number of physical delousing exposures by from 2 to 4 exposures. In various aspects, each targeted management method exposing the fish to the pest control agent compositions herein can reduce the number of physical delousing exposures by from 2 to 3 exposures. It will be appreciated that any reduction in the number of physical delousing exposures by the pest control agents herein will have an overall positive impact on the health and wellbeing of the fish, as well as an impact on the overall quality of flesh of the fish and a reduction in mortality in the fish population.

[0139] In some aspects of the methods herein, a first management method including an exposure to pest control agent can be immediately followed by a second management method including an exposure to a pest control agent, while in other aspects a first management method including an exposure can be followed by a second management method including an exposure to a pest control agent after waiting a predetermined period of time. The predetermined period of time can include from 1 day to 60 days, or any number of days falling within a range from 1 day to 60 days. In various aspects, the predetermined period of time can include more than 60 days. It will be appreciated that during the predetermined period of time, the fish exposed to pest control agent or a physical delousing system can be allowed to rest and recover from any external stresses that were placed upon them during either of the first or second management methods. In some aspects, the methods herein can include repeating a cycle of a first exposure to a pest control agent and a second exposure to a pest control agent for as long as necessary to reduce or prevent an infection or infestation with the pests described herein. In various aspects, a third exposure to a pest control agent, different than the first or second exposures to a pest control agent, can be used, where the third exposure to a pest control agent includes a concentration of pest control agent or an exposure to physical delousing system that is different than either of the first management method or second management method. [0140] The targeted management methods tailored to pest control agent concentration, targeted duration of time, and developmental life stage herein have surprisingly shown an effect on the extra daily mortality and extra mortality at molting when sea lice are examined at 2-months post infection after a 14-day exposure. These findings are detailed in the Examples herein. By way of example, administering a targeted concentration of azadirachtin-A rich fish feed or pest control agent composition can include administering a concentration from 0.01 mg/kg fish/day to 5.0 mg/kg fish/day for 14 days and then monitoring the fish over the course of one month or two months to determine efficacy of the targeted management methods on extra daily mortality and extra mortality at molting of the sea lice as compared to a population of lice within a fish population not fed a fish feed containing a pest control agent. Without wishing to be bound by any particular theory, it is believed that the efficacy of the targeted management methods, for both early life stages and later life stages, increases over time beyond the initial course of exposure due to the anti-feedancy properties of the pest control agent and the anti-molting properties of the pest control agent.

[0141] The targeted management methods herein can elicit a total reduction of lice present in an infection or infestation of a population of fish. The targeted management methods can produce an efficacy from a 90% reduction to a 98% reduction in the total number of sea lice present in an infection or infestation In various aspects, the targeted management methods using a concentration of pest control agent from 0.05 w/w % to 0.30 w/w % (i.e., approximately 1.5 mg/kg/day to 6.5 mg/kg/day) can produce an efficacy from a 95% to 99% reduction in the early life stages, including copepodid, chalimus 1, and chalimus 2 over a 14-day exposure time period. In various aspects, the targeted management methods using a concentration of pest control agent from 0.10 w/w % to 0.20 w/w % (i.e., approximately 2.5 mg/kg/day to 4.5 mg/kg/day) can produce an efficacy from a 91% to 98% reduction in the late chalimus 1, and chalimus 2 over a 14-day exposure time period.

Cage Systems

[0142] A number of cage systems have been developed to raise commercial fish for fish aquaculture. The cages can include sea cages deployed in the oceans off the coast of various countries such as Norway, Chile, Denmark, Scotland, and Canada, or the cages further can be deployed on land as land cages in various land-based aquaculture facilities. In various aspects, the cage systems can be fully or partially submerged in open water or freshwater bays, fjords, or inlets; can be moored to the ocean floor by one or more ropes, chains, anchors, and the like; can be suspended at the surface of the water by one or more floatation buoys; and can include one or more cages in the system; or any combination thereof. It will be appreciated that the cages discussed herein can be any suitable size, shape, and form to corral and maintain a population of farmed fish within the confines of the cage. The cages can be made from materials including netting, metal mesh, solid metal walls, polymeric mesh, solid polymeric walls, and the like.

[0143] Cage systems suitable for aquaculture can include open cage systems, enclosed cage systems, or semi-enclosed cage systems. As used herein, “open cage” refers to a cage that is at least partially submerged in open sea water or open freshwater and that retains farmed fishes therein in water that is in direct and unrestricted fluid communication with open sea water or open freshwater. In open cages, the water disposed within the open cage can be in constant flow with the currents and natural tides of water within the open sea water or open freshwater disposed on the exterior of the open cages. As used herein, “enclosed cage” refers to a cage that can be at least partially submerged in open sea water or open freshwater and that retains farmed fishes therein and that includes water that is not in fluid communication with any open sea water or open freshwater. In enclosed cages, the water disposed therein requires fresh seawater or fresh, freshwater to be continuously circulated through the enclosed cage system to provide oxygenation to the fish and to assist in removal of waste from the water contained within the enclosed cage. The water in an enclosed cage can utilize one or more barrier materials to control the exchange of water between the interior of the enclosed cage and the environment surrounding the cage. In various aspects, the water in enclosed sea cages further can be agitated with one or more streams of water, rotating mechanisms, and the like to simulate sea currents and water flow within the enclosed cage. It will be appreciated that in some aspects, enclosed cages are maintained at aquaculture facilities on land. As used herein, “semi-enclosed cage” refers to a cage that is at least partially submerged in open sea water or open freshwater and that retains farmed fishes therein in water that is in partial fluid communication with open sea water or open freshwater. In various aspects, the semi-enclosed cages can be in partial fluid communication with open sea water or open freshwater. In various aspects, partial fluid communication with the open seawater or open freshwater can include overtopping of water into the tank during high water currents, high tides, circulation of filtered fresh seawater or freshwater into or out of the cages, or during times of water filtration system failure.

Methods for Administering Pest Control Agents in Farmed Fish Ecosystems

[0144] The present disclosure provides methods for controlling or preventing parasitic infections or infestations in a farmed fish ecosystem. The methods include controlling or preventing parasitic infections or infestations in various types of cage systems utilized in farmed fish ecosystems. In various aspects the methods herein include administering pest control agents in fish feeds, pest control agent compositions, or both, to farmed fish contained inside one or more enclosed or semi-enclosed cage systems. In various additional aspects, the methods herein include administering pest control agents in fish feeds, pest control agent compositions, or both, to wild fish migrating into or out of the farmed fish ecosystems. It will be appreciated that multiple farmed fish ecosystems in a given geographical region can be managed with the methods herein in parallel or in series depending on the time of year. By way of example, the methods herein can be utilized in one or more farmed fish ecosystems during the migration of wild fish populations into and out of the farmed fish ecosystems. In some aspects, the methods herein include one or more management methods that include the administration of pest control agents to reduce the infection pressure within a given geographical region.

[0145] Without wishing to be limited to any particular theory, it is believed that the pest control agents, systems, and methods herein can result in a reduction in the total number of lice within any given geographical region that will aid to reduce the infection pressure within the region. The infection pressure can include the total number of sea lice at any given time in a given region and can further include internal infection pressure within a particular farm and external infection pressure originating from nearby farms. A reduction in infection pressure can be beneficial to both farmed and migrating wild fish populations by reducing, preventing, or controlling the infections and infestations within the farmed fish ecosystem. When the infection pressure is lowered farms are able to increase fish production. The increase in fish production, or an increase in fish biomass, increases total productivity of farms during a rearing cycle. A lowered infection pressure can further result in an increase in animal welfare by decreasing the amount of fish handling and exposure to pest control agents or physical delousing systems required over a course of the fish lifespan.

[0146] In an aspect, the present disclosure provides a method for controlling or preventing a parasitic infection or infestation in a farmed fish ecosystem by administering to farmed fish in one or more enclosed sea cages a fish feed including a pest control agent, the pest control agent including a neem extract rich in azadirachtin A. The methods herein further can include administering a pest control agent composition including a neem extract rich in azadirachtin A to the farmed fish before, during, or after administration of the fish feed. In various aspects, the methods herein further can include administering the fish feed upon introduction of fish into the one or more enclosed sea cages. In various aspects of the methods herein, the enclosed sea cages are fully enclosed sea cages or semi-enclosed sea cages. It will be appreciated that administering a fish feed supplemented with a pest control agent composition to fish can include feeding a fish feed supplemented with a pest control agent composition to fish. It will further be appreciated that administering a pest control agent composition to fish can include feeding a pest control agent composition to fish.

[0147] In an aspect, the present disclosure provides a method for controlling or preventing a parasitic infection or infestation in a farmed fish ecosystem by administering to one or more wild fish in the farmed fish ecosystem a first fish feed including a pest control agent, the pest control agent composition including a neem extract rich in azadirachtin A. The methods herein further can include administering to one or more farmed fish a second fish feed including a pest control agent, the pest control agent including a neem extract rich in azadirachtin A. The method further can include administering the second fish feed to one or more farmed fish at the same time as administering the first fish feed to one or more wild fish. The method further can include administering the second fish feed to one or more farmed fish at a different time than administering the first fish feed to one or more wild fish. In various aspects, the method further can include where the first fish feed is administered to one or more wild fish at the same concentration as the second fish feed that is administered to one or more farmed fish. In some aspects, the method further can include where the first fish feed is administered to one or more wild fish at a different concentration than the second fish feed that is administered to one or more farmed fish. The method further can include where the first fish feed and the second fish feed are administered at a concentration of Azadirachtin A from about 0.001 to 10.0 wt. % Azadirachtin A, and as described elsewhere herein. In certain aspects, the method further can include administering a pest control agent composition including a neem extract rich in azadirachtin A to the wild fish before, during, or after administration of the first fish feed. In other aspects, the method further can include administering a pest control agent composition comprising a neem extract rich in azadirachtin A to the farmed fish before, during, or after administration of the second fish feed.

[0148] The present disclosure provides a neem extract rich in azadirachtin A for reducing, preventing, or controlling a parasitic infection or infestation in a farmed fish ecosystem by following the steps including administering to farmed fish in one or more enclosed or semienclosed sea cages a fish feed comprising the neem extract rich in azadirachtin A.

[0149] It will be appreciated that in the various methods herein, the neem extract rich in azadirachtin A is not neem oil. [0150] The methods herein are directed to administering a fish feed or a pest control agent composition to one or more species of farmed fish belonging to one or more families including Cyprinidae, Cichlidae, Pangasiidae, Sciaenidae, Serranidae, Carangidae, Sparidae, Lateolabracidae, Moronidae, Mugilidae, Cypriniformes, Latidae, Eleotridae, Tilapiini, and Salmonidae. It will be appreciated that the fish feeds and pest control compositions herein can be administered to farmed or wile fish belonging to the Salmonidae family.

[0151] The methods described herein can manage a parasitic infection or infestation that is an ectoparasitic infection or infestation, or an endoparasitic infection or infestation in the enclosed sea cages. In various aspects, the methods herein are directed to management of a parasitic infection or infestation that includes a sea lice infection or infestation, or a copepod infection or infestation. The parasitic infection or infestation can include an infection or infestation with a copepod comprising one or more species of Caligus or Lepeophtheirus. In various aspects, the parasitic infection or infestation includes an infection or infestation with a copepod comprising one or more of Caligus demensi. Caligus rogercresseyi, or Lepeophtheirus salmonis.

[0152] It should be understood that the definitions described herein apply to all aspects as described unless otherwise stated.

[0153] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference is to be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0154] Values expressed in a range format are to be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1 % to about 5 %” or “about 0.1 % to 5 %” is to be interpreted to include not just about 0.1 % to about 5 %, but also the individual values (e.g., 1 %, 2 %, 3 %, and 4 %) and the sub-ranges (e.g., 0.1 % to 0.5 %, 1.1 % to 2.2 %, 3.3 % to 4.4 %) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise. [0155] Unless expressly stated, ppm (parts per million), percentage, and ratios are on a by weight basis. Percentage on a by weight basis (% w/w or w/w %) is also referred to as weight percent (wt. %) or percent by weight (% wt.) herein.

[0156] Exemplary embodiments of the present invention are as follows:

[0157] Embodiment 1 : A method for reducing, preventing, or controlling a parasitic infection or infestation in a farmed fish ecosystem comprising: administering to farmed fish in one or more enclosed or semi-enclosed sea cages a fish feed comprising a pest control agent, the pest control agent comprising a neem extract rich in azadirachtin A.

[0158] Embodiment 2: The method of Embodiment 1, wherein the neem extract rich in azadirachtin A comprises from 15 wt. % to 33 wt. % azadirachtin A.

[0159] Embodiment 3: The method of any of Embodiments 1-2, wherein the fish feed is administered upon introduction of fish into the enclosed sea cages.

[0160] Embodiment 4: The method of any of Embodiments 1-2, wherein the fish feed is administered for a period of time prior to introduction of fish into the enclosed sea cages.

[0161] Embodiment 5: The method of any of Embodiments 1-4, wherein the enclosed sea cages are fully enclosed sea cages or semi-enclosed sea cages.

[0162] Embodiment 6: The method of any of Embodiments 1-5, wherein the fish feed is administered to the fish for at least 14 days.

[0163] Embodiment 7: The method of any of Embodiments 1-6, wherein the neem extract rich in azadirachtin A is administered to the fish at a concentration from 1.5 mg to 2.5 mg azadirachtin A per kg body weight per day.

[0164] Embodiment 8: The method of any of Embodiments 1-6, wherein the neem extract rich in azadirachtin A is administered to the fish at a concentration from 2.6 mg to 5.0 mg azadirachtin A per kg body weight per day.

[0165] Embodiment 9: The method of any of Embodiments 1-8, further comprising administering a second fish feed comprising a pest control agent comprising a neem extract rich in azadirachtin A to wild fish within the farmed fish ecosystem.

[0166] Embodiment 10: The method of any of Embodiments 1-9, wherein the parasitic infection or infestation is an ectoparasitic infection or infestation, or an endoparasitic infection or infestation.

[0167] Embodiment 11 : The method of any of Embodiments 1-10, wherein the parasitic infection or infestation is a sea lice infection or infestation, or a copepod infection or infestation. [0168] Embodiment 12: The method of any of Embodiments 1-11, wherein the parasitic infection or infestation comprises an infection or infestation with a copepod comprising one or more species of Caligus or Lepeophtheirus .

[0169] Embodiment 13: The method of any of Embodiments 1-12, wherein the parasitic infection or infestation comprises an infection or infestation with a copepod comprising one or more of Caligus clemensi, Caligus elongatus, Caligus rogercresseyi, o Lepeophtheirus salmonis. [0170] Embodiment 14: The method of any of Embodiments 1-13, further comprising administering a pest control agent composition to the farmed fish before, during, or after administration of the fish feed.

[0171] Embodiment 15: The method of any of Embodiments 1-14, wherein the neem extract does not comprise neem oil.

[0172] Embodiment 16: The method of any of Embodiments 1-15, wherein the neem extract rich in azadirachtin A is obtained by a method comprising the steps of: providing neem seeds; crushing the neem seeds; extracting azadirachtin from the crushed seeds with water; adding a second extraction solution that comprises: a non-aqueous solvent which is not miscible with water and has a higher solubility of azadirachtin than water; or a surfactant having a turbidity temperature between 20 °C and 80 °C; and recovering the concentrated azadirachtin from the second extraction solution.

[0173] Embodiment 17: The method of any of Embodiments 1-16, wherein the fish feed further comprises one or more components comprising antibiotic agents, antibacterial agents, antifungal agents, antiviral agents, antiparasitic agents, or antiprotozoal agents.

[0174] Embodiment 18: The method of any of Embodiments 1-17, wherein the fish feed is administered to species of farmed fish belonging to one or more families comprising Cyprinidae, Cichlidae, Pangasiidae, Sciaenidae, Serranidae, Carangidae, Sparidae, Lateolabracidae, Moronidae, Mugilidae, Cypriniformes, Latidae, Eleotridae, Tilapiini, and Salmonidae.

[0175] Embodiment 19: A method for reducing, preventing, or controlling a parasitic infection or infestation in a farmed fish ecosystem comprising: administering to wild fish present in the farmed fish ecosystem a first fish feed comprising a pest control agent, the pest control agent comprising a neem extract rich in azadirachtin A. [0176] Embodiment 20: The method of Embodiment 19, wherein the neem extract rich in azadirachtin A comprises from 15 wt. % to 33 wt. % azadirachtin A.

[0177] Embodiment 21 : The method of any of Embodiments 19 or 20, further comprising administering to farmed fish within the farmed fish ecosystem a second fish feed comprising a pest control agent, the pest control agent comprising a neem extract rich in azadirachtin A.

[0178] Embodiment 22: The method of Embodiment 21, further comprising administering the second fish feed to the farmed fish at the same time as administering the first fish feed to the wild fish.

[0179] Embodiment 23 : The method of Embodiment 22, further comprising administering the second fish feed to the farmed fish at a different time than administering the first fish feed to the wild fish.

[0180] Embodiment 24: The method of any of Embodiments 19-23, wherein the neem extract rich in azadirachtin A in the first fish feed is administered to the wild fish at the same concentration as the neem extract rich in azadirachtin A in the second fish feed that is administered to the farmed fish.

[0181] Embodiment 25: The method of any of Embodiments 19-23, wherein the first fish feed is administered to the wild fish having a different concentration of neem extract rich in azadirachtin A than the neem extract rich in azadirachtin A in the second fish feed that is administered to the farmed fish.

[0182] Embodiment 26: The method of any of Embodiments 19-25, wherein the first fish feed and the second fish feed are administered at a concentration of azadirachtin A from about 0.001 wt. % to 10.0 wt. % azadirachtin A.

[0183] Embodiment 27: The method of any of Embodiments 19-26, wherein the parasitic infection or infestation is an ectoparasitic infection or infestation, or an endoparasitic infection or infestation.

[0184] Embodiment 28: The method of any of Embodiments 19-27, wherein the parasitic infection or infestation is a sea lice infection or infestation, or a copepod infection or infestation. [0185] Embodiment 29: The method of any of Embodiments 19-28, wherein the neem extract rich in azadirachtin A is obtained by a method comprising the steps of: providing neem seeds; crushing the neem seeds; extracting azadirachtin from the crushed seeds with water; adding a second extraction solution that comprises: a non-aqueous solvent which is not miscible with water and has a higher solubility of azadirachtin than water; or a surfactant having a turbidity temperature between 20 °C and 80 °C; and recovering the concentrated azadirachtin from the second extraction solution.

[0186] Embodiment 30: The method of any of Embodiments 19-29, wherein the neem extract rich in azadirachtin A does not comprise neem oil.

[0187] Embodiment 31 : The method of any of Embodiments 19-30, wherein the fish feed further comprises one or more components comprising antibiotic agents, antibacterial agents, antifungal agents, antiviral agents, antiparasitic agents, or antiprotozoal agents.

[0188] Embodiment 32: The method of any of Embodiments 19-31, wherein the fish feed is administered to any species of wild fish belonging to one or more families comprising Cyprinidae; Cichlidae; Pangasiidae; Sciaenidae; Serranidae; Carangidae; Sparidae; Lateolabracidae; Moronidae; Mugilidae; Cypriniformes; Latidae; Eleotridae; Tilapiini and Salmonidae.

[0189] Embodiment 33: The method of any of Embodiments 19-32, wherein the parasitic infection comprises an infection or infestation with a copepod comprising one or more species of Caligus or Lepeophtheirus.

[0190] Embodiment 34: The method of any of Embodiments 19-33, wherein the ectoparasitic infection comprises an infection or infestation with a copepod comprising one or more of Caligus clemensi, Caligus elongatus, Caligus rogercresseyi, or Lepeophtheirus salmonis.

[0191] Embodiment 35: The method of any of Embodiments 19-34, further comprising administering a pest control agent composition comprising a neem extract rich in azadirachtin A to the farmed fish or the wild fish before, during, or after administration of the first fish feed.

[0192] Embodiment 36: A neem extract rich in azadirachtin A for reducing, preventing, or controlling a parasitic infection or infestation in a farmed fish ecosystem by following the steps comprising: administering to farmed fish in one or more enclosed or semi-enclosed sea cages a fish feed comprising the neem extract rich in azadirachtin A.