Compositions and methods for managing non-copepods parasitic disease in aquaculture systems

Abstract

The present disclosure provides a method for reducing, preventing, or controlling non-copepods parasitic infection or infestation in a fish school. The method comprises providing a fish feed comprising an Azadirachtin A-rich Azadirachtin A extract, the Azadirachtin A-rich Azadirachtin A-rich Azadirachtin A-rich Azadirachtin A-rich Azadirachtin A-rich Azadirachtin A-rich Azadirachtin A-rich Azadirachtin A-rich Azadirachtin A-rich Azadirachtin A-rich Azadirachtin A-rich wherein the fish feed provides a concentration of 0.01 mg to 5.0 mg azadirachtin A / kg body weight / day to the one or more fish. Other aspects are also provided herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 381,369, filed on October 28, 2022, and entitled “COMPOSITIONS AND METHODS FOR THE MANAGEMENT OF PARASITIC DISEASE IN AQUACULTURE SYSTEMS,” which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to compositions and methods for managing parasitic diseases in fish. In particular, the present disclosure relates to compositions and methods for managing non-copepod parasite infections or infestations in fish populations by applying pest control agents via fish feed or pest control agent compositions. Background Art

[0004] There are a variety of harmful parasites that can target fish in aquaculture and can cause immune response disorders in fish during infection or infestation. Immune system disorders can include suppressing protective, beneficial immune and physiological responses while exacerbating non-protective or harmful immune responses. This disorder can cause a negative immune response, whereby the immune system may be stopped, blocked, or reduced in effectiveness when faced with an immune attack. In addition, the overactivation of non-protective immune responses can directly damage the host's tissues, which can lead to immunopathology in the host. For example, salmon lice (Lepeopththeirus salmonis) is an ectoparasite that has adapted to attach to salmon during evolution and remains unharmed by the immune system of the fish. Without wishing to be bound by any particular theory, it is believed that salmon lice can regulate and escape host immunity by secreting one or more molecules (including proteins, lipids) to induce inhibitory effects on protective, beneficial immune responses to fish. The downregulation of this protective host response can lead to opportunistic infections of other parasites, germs, viruses, protozoa, amoebas, etc., which in turn may lead to the upregulation of non-protective or harmful immune responses. Many viral parasites modulate the host immune response to create an environment that is conducive to the progression of infection. In some cases, downregulation of a protective host response can itself lead to upregulation of a non-protective or harmful immune response. In addition, overstimulation of a non-protective or harmful immune response can produce many adverse effects on fish at the physiological, histological, biochemical and enzymatic levels. For example, the energy cost of a non-protective immune or physiological response may interfere with the ability of fish to clear toxic compounds, handle oxidative stress, heat stress or other environmental challenges.

[0005] Various viruses and bacteria known to infect fish are thought to cause upregulation of non-protective or deleterious immune responses. Upregulation can lead to activation or overactivation of the host immune response in fish and can result in undesirable inflammatory side effects that can cause tissue damage (i.e., immunopathology), reduced animal welfare, farm productivity loss, and even fish mortality. For example, Ichthyophthirius multifiliis can infect salmon and cause significant economic losses to fish farmers.

[0006] There are a variety of mechanisms for managing parasitic diseases, including various synthetic agents, pickling techniques, and vaccines. Such agents may be difficult to implement in the long term and are expensive. In addition, resistance to various agents remains a problem in the art. Therefore, there is a need to provide easy-to-use management methods that can be applied to fish to manage non-copepod parasitic diseases by using simple and cost-effective means. Summary of the invention

[0007] The present disclosure provides a method for reducing, preventing or controlling a non-copepod parasite infection or infestation in a fish population, the method comprising providing a fish feed comprising a neem extract enriched in azadirachtin A, the neem extract comprising 15 wt% to 33 wt% azadirachtin A, and administering the fish feed comprising the neem extract enriched in azadirachtin A to one or more fish, wherein the fish feed provides a concentration of 0.01 mg to 5.0 mg azadirachtin A / kg body weight / day to the one or more fish.

[0008] In one aspect, the method further comprises wherein the non-copepod parasite infection or infestation is caused by one or more parasites comprising Amyloodinium ocellatum, Cardicola forsteri, Cryptocaryon irritans, Diplectanum spp., Gyrodactylus salaris, Henneguya salminicola, Ichthyophthirius multifida, Kudoa thyrsites, Myxobolus cerebralis, Neoparamoeba perurans, Saprolegnia parasitica, Sparicotyle chrysophrii, Trichodina spp., or Zeylanicobdella arugamica. arugamensis).

[0009] In one aspect, the method further includes wherein the fish feed is administered to the farmed fish for at least 11 days.

[0010] In one aspect, the method further includes wherein the fish feed is administered to farmed fish for at least 14 days.

[0011] In one aspect, the method further includes wherein the concentration of azadirachtin A comprises a concentration in an amount effective to increase the efficacy of azadirachtin A against non-copepod parasitic infection or infestation compared to fish fed a diet lacking the neem extract enriched in azadirachtin A.

[0012] In one aspect, the method further comprises wherein the neem extract enriched in azadirachtin A is administered to the fish at a concentration of 1.5 mg to 2.5 mg azadirachtin A / kg body weight / day.

[0013] In one aspect, the method further comprises wherein the neem extract enriched in azadirachtin A is administered to the fish at a concentration of 2.6 mg to 5.0 mg azadirachtin A / kg body weight / day.

[0014] In one aspect, the method also includes wherein the fish feed further comprises one or more components including an antibacterial, antifungal, antiviral, antiparasitic, or antiprotozoal agent.

[0015] In one aspect, the method also includes wherein the fish feed is administered to fish species belonging to one or more families including: Cyprinidae, Cichlidae, Panfish, Sciaenidae, Serranidae, Carangidae, Sparidae, Perchidae, Lycaenidae, Mugilidae, Cypriniformes, Percidae, Channaidae, Tilapia and Salmonidae.

[0016] In one aspect, the method further includes wherein the pest control agent is configured to produce an inhibitory effect comprising one or more of an antiparasitic effect, an antibacterial effect, an antiviral effect, an antifungal effect, or an antiprotozoal effect.

[0017] In one aspect, the method further includes wherein the neem extract enriched in azadirachtin A does not comprise neem oil.

[0018] In one aspect, the method further includes wherein the pest control agent is provided to the pest in the fish feed in an amount sufficient to modify the behavior of the pest.

[0019] In one aspect, the method also includes wherein modulating the behavior of the pest comprises one or more of: a change in feeding habits, a change in feeding patterns, a change in appetite, a change in movement patterns, or a change in mating patterns compared to pests found on control animals not fed the pest control agent.

[0020] In one aspect, the method also includes where the change in eating pattern can include a decrease in eating pattern.

[0021] In one aspect, the method also includes wherein the alteration in appetite can include a decrease in appetite.

[0022] In one aspect, the method also includes where the change in movement can include a decrease in movement.

[0023] In one aspect, the method also includes where the alteration in mating pattern can include a decrease in mating pattern.

[0024] In one aspect, the method also includes where the alteration in mating patterns can include a reduction in the number of live pests present on the fish or in the fish's habitat as compared to pests found on control fish not fed the pest control agent.

[0025] In one aspect, the method also includes wherein a neem extract enriched 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 solvent comprising a non-aqueous solvent that is immiscible 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 concentrated azadirachtin from the second extraction solution.

[0026] The present disclosure provides a method for reducing, preventing or controlling a non-copepod parasite infection or infestation in fish, the method comprising: providing a pest control agent composition comprising a pest control agent comprising a neem extract enriched in azadirachtin A; and applying the pest control agent composition to one or more fish during an infection or infestation for 1 day to 20 days, wherein the concentration of azadirachtin A applied to the fish by the pest control agent composition is 0.01 mg to 5 mg azadirachtin A / kg body weight / day. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] not applicable. DETAILED DESCRIPTION

[0028] Reference will now be made in detail to certain aspects of the disclosed subject matter.While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0029] The present disclosure provides fish feed and pest control agent compositions, which contain one or more pest control agents for reducing, preventing or controlling parasitic infections or infestations in fish or fish schools. Various pests can cause disorders of protective immune responses or protective physiological responses in host fish, and they can regulate immune responses and physiological responses to benefit them. Some parasitic species can benefit by exposing the host to anti-inflammatory mediators to suppress the host's protective immune response or protective physiological response. Some parasitic species benefit by stimulating non-protective immunity and non-protective physiological responses (which in turn suppress the activation of protective immunity and protective physiological responses). Some parasitic species can impose regulatory pressure on host fish for a sufficiently long time, so that the immune response of the host fish produces harmful immunomodulatory or inflammatory effects on the fish for a long time.

[0030] Fish conditioned to parasite dominance are less likely to be able to activate a protective immune response or a protective physiological response, such as a Type 1 immune pathway. For example, the presence of some parasites, such as sea lice, can induce the fish to produce antibodies. However, the antibodies produced do not confer immune protection to the host, and can actually deplete the host's valuable resources, thereby facilitating the provision of a suitable environment for sea lice. Similarly, some parasites can upregulate the production of protective mucus found on the surface of the fish's skin. Salmon produce mucus as a physical and chemical protective barrier against invading pathogens. Certain parasites can take advantage of this protective physiological response by upregulating the volume of mucus produced by the fish, which in turn can provide a rich food source for the parasites.

[0031] The pest control agent herein can be applied to fish, and can effectively regulate the immune response or physiological response of the host fish in the presence or absence of parasites. The regulation of immune response or physiological response can be applied by the pest control agent before, after or during infection or infestation. The regulation of immune response or physiological response can occur by reducing the imbalance of immune response or physiological response caused by various parasites during infection or infestation. In some aspects, the pest control agent can stimulate the protective immune response in fish or suppress the harmful immune response. By stimulating the protective immune response and suppressing the harmful immune response, the pest control agent herein can directly or indirectly help to reduce, prevent or control the infection or infestation caused by pests described herein. In some aspects, the pest control agent herein can have a direct antiparasitic effect on the parasites that feed on the host that has ingested the pest control agent, thereby reducing the ability of the parasite to immunoregulate its host. The pest control agent can induce immunomodulatory effects, and by alleviating the parasite load in the fish or fish school, the immune response and physiological response of the host can be restored to the state before the imbalance, thereby providing an anti-inflammatory effect. In addition, pest control agents can act on host immune and physiological pathways that can contribute to the positive outcome of pest infection or infestation. For example, pest control agents can promote effective antimicrobial immune responses, inhibit non-protective and harmful immune responses, stimulate antioxidant defenses, detoxification pathways, repair tissue damage mediated by autoimmune responses or caused by pathogens, and promote wound repair.

[0032] Regulation of the immune response or physiological response in the host fish by the pest control agent may include various immunomodulatory effects in the fish. Immunomodulatory effects may include reducing or preventing various immunopathologies caused by an immune response disorder of the physiological response. Immunopathologies may include tissue damage caused by an excessive immune response, such as reducing myocardial damage, skeletal muscle damage, etc.

[0033] The present disclosure provides fish feed, pest control agent compositions and pest control agents for reducing, preventing or controlling one or more non-copepod parasites in fish and fish populations. As used herein, the term "copepod" refers to a group of crustaceans found in freshwater and in marine waters and having one or more parasitic stages of their life cycle, including various sea lice species. As used herein, the term "non-copepod" refers to a group of parasites that are not copepods and are known to infect or infest fish species that are not copepods.

[0034] As used herein, the term "infection" may refer to a condition in which pests (including various pests as defined elsewhere herein) can invade any internal or external part of a host organism, causing damage to the host organism, and the pests use components of the host organism to maintain themselves, reproduce or colonize the host organism.

[0035] As used herein, the term "infestation" may refer to the presence of an abnormally large number of pests as defined herein, wherein the pests are concentrated in an area in numbers that can cause damage or disease by infecting host organisms.

[0036] As used herein, the term "pest" may refer to any organism that is detrimental to the health, value, or appearance of another organism. The term pest may include, but is not limited to, one or more of various parasites, including worms, helminths, and trematodes; one or more bacteria; one or more viruses; one or more types of fungi; and various protozoa (e.g., amoebas).

[0037] As used herein, the term "parasite" may refer to one or more external parasites or internal parasites. As used herein, the term "internal parasite" may refer to an organism that inhabits one or more internal microhabitats of another organism. For example, an internal parasite may inhabit one or more of a tissue, an organ, or a system of a host organism. For example, an internal parasite may inhabit the intestines, blood, or both of a host organism. As used herein, the term "external parasite" may refer to an organism that inhabits an external microhabitat of another species or occupies an external microhabitat of another species. For example, an external parasite may inhabit or occupy the surface of a host species. In the case of fish, external parasites may inhabit the skin of the fish, where they sometimes stay between scales, and they may also feed on mucus, blood, skin, gills, muscle, or any combination thereof.

[0038] As used herein, the term "pest control agent" may refer to an agent used to reduce, prevent or control an infection or infestation caused or contributed to by one or more pests. In various aspects, the pest control agent described herein may refer to an agent used to reduce, prevent or control an infection or infestation caused by one or more endoparasites or ectoparasites.

[0039] Target fish

[0040] Fish feed provided herein can be fed as a fish feed diet, or used to feed any fish susceptible to infection or infestation by one or more pests. For example, fish feed can be used as a component of a diet fed to any farmed fish (including, for example, commercially relevant fish species) in aquaculture. For example, fish feed provided herein can form a part of a diet fed to any freshwater fish, saltwater fish, or marine fish. Fish feed can be used as a component of a diet fed to any species belonging to the following families: Cyprinidae, Cichlidae, Catfish, Sciaenidae, Serranidae, Carpidae, Sparidae, Perchidae, Lysimachia, Mugilidae, Cypriniformes, Perchidae, Channaidae, Tilapia, and Salmonidae. Therefore, the fish feed herein can be used to feed species belonging to any genus within these families, particularly those species that are cultured for human or animal consumption. For example, the fish feed described herein can be used to feed species belonging to Salmo and / or Oncorhynchus, but is not limited thereto. In particular, the fish feed 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 salar), Pacific salmon, Arctic char (Char) or rainbow trout. In addition, 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 for the pet and ornamental fish industry, for example, for pest control of koi (Cyprinus rubrofuscus) and goldfish (Carassiusauratus).

[0041] Target parasites

[0042] Pests of target fish herein may include non-copepod parasites that can cause a variety of diseases in fish. Non-copepod parasites suitable for management with the pest control agents described herein may include one or more of the following: Oval Flagellate, Heart Fluke, Cryptocaryon Irritans, Species of Scale Plate, Trichoderma salmonis, Salmonid Myxosporea, Multi-sperm Ichthyophthirius, Kudao of Snake Mackerel, Brain Iodine Bubble, New Parameba, Saprolegnia species (including parasitic Saprolegnia), Monogenea, Trichodina species or Arugam Ceylon leech. These organisms can all cause one or more clinical manifestations of the disease, which reduce the health of the fish, affect the edible muscle quality of the fish, can reduce farm productivity by reducing the amount of fish on the market, increase the mortality rate of the fish, and can cause significant economic losses to the farmers.

[0043] Dinoflagellate ectoparasites (flagellates) are known to cause amyloodiniosis in farmed and aquarium fish. The main feature of amyloodiniosis is infestation of the fish's gills, but infestations of the skin, fins, and eyes may also occur. Clinical signs of amyloodiniosis may include loss of appetite, decreased activity, labored breathing, and general poor health of the fish.

[0044] The parasitic ciliate (Ichthyophthirius multifida) can cause a fish disease commonly known as "Ich". Ichthyophthirius multifida shows low host specificity and infests the interior of the organism on the epithelial layer of the gills, skin and fins. Ichthyophthirius multifida can cause significant damage to the skin, gills and fins of its host, and the fish will experience pain as a result, and cause anorexia, breathing difficulties, discoloration and mottling, inactivity, scratching objects and swimming upside down. If not controlled, Ichthyophthirius multifida can cause 100% mortality.

[0045] The ciliate Cryptocaryon irritans is known to cause marine white spot disease in both wild and farm-raised fish. The Cryptocaryon irritans organism feeds on the skin, eyes, and gills of fish and can degrade the function of these tissues. Marine white spot disease caused by Cryptocaryon irritans can cause white nodules to appear on the fish and can lead to excessive mucus production, skin discoloration, loss of appetite, hyperactivity, poor general health, and breathing difficulties.

[0046] The organisms of the genus Diplectanum are parasites that infect the gills of fish species. Those Diplectanum suitable as targets of the pest control agents herein may include, but are not limited to, Diplectanum aculeatum, Diplectanum aequans, Diplectanum banyulense, Diplectanum belengeri, Diplectanum bocqueti, Diplectanum chabaudi, Diplectanum copiosum, Diplectanum dollfusi, Diplectanum femineum, or Diplectanum flagritubus. Diplectanum species may infest fish by feeding on gills and causing diplectanosis in fish. Fish with ichthyosis may suffer from damaged gill tissue, difficulty breathing, poor overall health, and secondary infections due to viruses and bacteria.

[0047] The salmon fluke (Trichodina salmonis) is a parasitic species that lives on the body surface of many freshwater fish. Trichodina salmonis feeds on the body of many freshwater fish and can cause skin wounds and expose the fish to co-infection with one or more additional pests.

[0048] The heart fluke is a trematode that infects bluefin tuna. It can cause severe gillitis, or swelling of the gills, and myocarditis in fish, and can lead to high mortality in fish stocks.

[0049] Kudaoa serrata is a myxozoan parasite that infects the muscles of a wide range of marine fish and is distributed throughout the world. Kudaoa serrata can cause nodules or cysts in the muscle tissue of fish. The infection can lead to an excessive immune response that can cause tissue degeneration, whereby the tissue becomes soft due to enzymatic digestion of the muscle fibers. Fish infected with Kudaoa serrata may be damaged to the point where they are no longer of sufficient quality to be sold to consumers, resulting in significant losses to the fish farmers.

[0050] The myxosporean Henneguya zschokkei can infect or infest fish and cause a disease known as milky meat disease or cassava disease. Milky meat disease or cassava disease caused by Henneguya zschokkei is characterized by the intermittent appearance of small white cysts filled with spores of the organism on the muscles of the fish. Milky meat disease can destroy farm productivity because, although the disease is generally harmless, it can result in a loss of economic profit due to the unsightly appearance of the fish.

[0051] The myxosporean cephalosporin causes vertigo in various fish species. Vertigo can be characterized as a chronic disease that infects salmon, especially juvenile salmon, and causes severe deformation of the fish's cartilage, bones, and muscles. When the parasite infects the cartilage of fish, its presence can put pressure on the spinal cord and cause a characteristic swimming pattern in the form of a whirlpool. The disease can be extremely uncomfortable for fish and can cause serious economic losses to fish farmers.

[0052] The protozoan Paramoeba perurans (synonym, Paramoeba perurans) causes amoebic gill disease in various fish species. The protozoan attaches to the gills of the fish and causes tissue damage through increased mucus production and multifocal plaques of swollen tissue. The fish experience breathing difficulties due to gill damage. The disease causes extreme pain to the fish and can cause serious economic losses to fish farms.

[0053] Biomonogenes is a parasite that infects the gills of various fish species. Once the organism attaches to the gills, it causes tissue damage that can lead to breathing difficulties, anemia, and death. The disease causes extreme pain in the fish and can cause severe economic losses to fish farms.

[0054] Single-celled flagellates of the genus Trichodina can infect or infest a variety of fish, including those commonly found in ornamental fish ponds, such as koi and goldfish. The trichodina attach to the fish's skin and gills and cause damage to these tissues as well as breathing difficulties, loss of appetite, and loss of scales.

[0055] Fungi of the genus Saprolegnia, including Saprolegnia parasiticus, can infect or infest various fish types and can cause saprolegniasis. In fish, saprolegniasis is characterized by the presence of grayish-white patches with filamentous hyphae on the body or fins of the fish. The condition can cause tissue damage and, in extreme cases, death.

[0056] The Arugam ceylonis is a marine leech that can parasitize many fish species raised in aquaculture. The Arugam ceylonis leech attaches to the skin, gills, fins, and oral surfaces of various fish species and causes wounds in the area of ​​attachment. Wounds caused by attachment and feeding by the Arugam ceylonis leech can predispose the fish to secondary infections by other pests.

[0057] Fish feed

[0058] The present disclosure provides fish feed used as fish diet in aquaculture applications.It should be understood that the fish feed herein can include fish feed of the appropriate type specific to a given fish species.The fish feed can be used as a component of the diet of any species belonging to the following families: Cyprinidae, Cichlidae, Panfish, Sciaenidae, Serranidae, Carangidae, Sparidae, Perchidae, Lysimachia, Mugilidae, Cypriniformes, Perchidae, Channaidae, Tilapia and Salmonidae.In various fields, the present disclosure provides a kind of fish feed or fish feed diet for the species in Salmonidae.The fish feed provided herein can be used for feeding wild fish or cultured fish.In various fields, both wild fish and cultured fish can be fed simultaneously.Further, the fish feed can be used for feeding freshwater fish or saltwater (e.g., seawater) fish or both.

[0059] Fish feed of the present disclosure can be produced using raw materials as a basal feed formulation as a solid feed or a liquid feed, which raw materials can be selected based on its application and 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 may include both solid fish feed components and liquid fish feed components. The fish feed in solid form may include particles, extruded blocks, steam particles, flakes, tablets, powders, etc. In various aspects, the basal feed may include basal feed particles. In some aspects, the basal feed particles may include a porous matrix distributed throughout. The fish feed in liquid form may include an aqueous solution, an oil, an oil and water emulsion, a slurry, a suspension, etc. In various aspects, the solid fish feed may also include one or more oils, which are arranged on the surface or distributed throughout the fish feed.

[0060] The fish feed herein may include a variety of different ingredients or raw materials that can maintain the life, growth and reproduction of fish. The fish feed may include any substrate that is edible to fish. For example, an edible substrate may provide a source of nutrition for fish, or may be an inert substrate that has no nutritional value to fish. In various aspects, the fish feed herein may include feed as a nutritional fish feed or a non-nutritional feed. The nutritional fish feed may include a nutritional food formulated for fish as part of its diet as the main source of nutrition, growth and reproduction. Suitable nutritional fish feeds may include one or more of the following: proteinaceous substances as a source of protein, peptides and amino acids; carbohydrates; and fats, as described below. Non-nutritional fish feeds may include any substrate that is edible to fish but does not provide nutrition to maintain life, growth or reproduction. In various aspects, the nutritional or non-nutritional fish feed herein may include one or more compounds intended to change the quality, quantity or appearance of fish and fish tissues. For example, nutritional or non-nutritional fish feeds may include carotenoid compounds to improve the appearance (e.g., color) of muscle tissue. For example, carotenoid compounds may include compounds such as astaxanthin.

[0061] Fish feeds herein may include complete fish feeds. Complete fish feeds may include feeds for fish that are compounded to feed as the sole ration and can maintain life, promote growth and maintain reproduction without taking in any additional substances except water. Complete fish feeds may include a complex mixture containing various energy sources such as carbohydrates, proteins and fats. In various aspects, fish feeds herein may include at least protein and starch. Additional ingredients necessary to support the life, growth and reproduction of fish, such as vitamins and minerals, may be included. 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, starch, tapioca starch, wheat, wheat gluten, guar meal, guar protein concentrate, peas, pea protein concentrate, pea starch, beans, broad beans, sunflower meal, vegetable oil, canola oil, poultry oil, rapeseed oil, fish oil, soybean oil, hydro-pressed linseed oil, camelina oil, lecithin, macromolecular minerals, minerals, vitamins, amino acids, pigments, and any combination thereof. It should be understood that the fish feed herein can include fish meal, which can include materials of plant or animal origin. Any animal-derived materials present in the fish meal can be derived from fish of the same species or fish of a different species (e.g., heterologous species or non-fish species).

[0062] The total protein content in the fish feed can be 10% to 70% by weight, 15% to 65% by weight, 20% to 60% by weight, or 25% to about 55% by weight. 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 any of the foregoing ranges. The total protein in the fish feed can vary depending on the formulation, species, age, and intended use of the feed. It should be understood that the various protein requirements of the species of fish receiving the fish feed can be adjusted to meet the protein requirements of the species.

[0063] The protein in the fish feed herein can come from any suitable source, including but not limited to one or more of the following: fish meal, terrestrial animal protein (e.g., poultry meal), plant-based protein (e.g., vegetable meal), or any combination thereof. The fish feed can contain 0% to 80%, 10% to 80%, 20% to 75%, 30% to 70%, 60% to 80%, or 10% to 30%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 75% fish meal by weight, or any amount within any of the foregoing ranges. The fish feed may comprise 0% to 80%, 10% to 80%, 20% to 75%, 30% to 70%, 60% to 80%, or 10% to 30%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 75% by weight of terrestrial animal protein, or any amount within any of the foregoing ranges. The fish feed may comprise between 0% to 80%, 10% to 80%, 20% to 75%, 30% to 70%, 60% to 80%, or 10% to 30%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 75% by weight of plant-based protein, or any amount within any of the foregoing ranges.

[0064] The total fat (e.g., oil, fat and / or lipid) in the fish feed can be 5% to 50%, 10% to 45%, 15% to 40% or 20% to 35%. The total fat in the fish feed can be at least 5% by weight, 10% by weight, 15% by weight, 20% by weight, 25% by weight, 30% by weight, 35% by weight, 40% by weight, 45% by weight or at least 50% by weight, or any amount within any of the foregoing ranges. The total fat in the fish feed can vary depending on the formulation, the target fish species and the intended use of the feed. It should be understood that the various requirements of the species of fish receiving the fish feed for fat can be adjusted to meet the requirements of the species for fat. Fats suitable for use herein can include, but are not limited to, fats provided by canola oil, poultry oil, rapeseed oil, fish oil, soybean oil, hydro-pressed linseed oil, camelina oil, palm oil, lecithin or any combination or fraction thereof.

[0065] The moisture content of the fish feed herein can vary depending on the content and preparation method of the feed. In various aspects, the moisture content can be 1 wt % to 20 wt %, 2 wt % to 18 wt %, 5 wt % to 15 wt % or 6 wt % to 12 wt %.

[0066] The fish feed herein may include one or more pest control agents. In various aspects, the one or more pest control agents may be present in the fish feed in an amount effective to produce an inhibitory effect on one or more pests, as described elsewhere herein. Therefore, the fish feed herein may include a pest control agent for preventing and treating an infection or infestation caused by one or more pests. In various aspects, the fish feed herein may include a pest control agent for preventing and treating an infection or infestation of an endoparasite or an ectoparasite. Various pests suitable as targets of the pest control agents herein are described elsewhere.

[0067] Each pest control agent contained in the fish feed herein may be individually capable of controlling one or more of parasitic, bacterial, viral, fungal or protozoan infections or infestations. Therefore, it should be understood that any given pest control agent used in many aspects described herein may be referred to as exhibiting one or more inhibitory effects, including antiparasitic effects (e.g., anti-ectoparasites, anti-endoparasites), antibacterial effects, antiviral effects, antifungal effects, or antiprotozoal effects. In various aspects, the inhibitory effect may include an antiparasitic effect, wherein the antiparasitic effect may also include an anti-ectoparasitic effect, an anti-endoparasitic effect, or both. It should be understood that the inhibitory effect may result in reducing, preventing or controlling the concentration and spread of various parasites, bacteria, viruses, fungi or protozoan organisms described herein. In various aspects, the pest control agents of the present disclosure may produce an inhibitory effect on 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 parasites may include completely preventing infection or infestation in a fish population or on each individual fish, reducing the total number of parasites present in a fish population or on each individual fish, or controlling how many parasites are present in a fish population or on each individual fish in accordance with local regulatory requirements. It should be understood that the inhibitory effects described herein may be measured on a population of fish infected or infested with one or more pests that is fed a diet lacking azadirachtin A-enriched neem extract.

[0068] In other aspects, the inhibitory effect on pests may include one or more of anti-feeding, anti-molting, anti-fertility and anti-reproductive effects or anti-parasitic effects. As used herein, "anti-feeding" may refer to the effect of one or more pest control agents in stopping or inhibiting pest feeding, thereby causing malnutrition, stunted development, molting obstruction or delay, and death. Malnourished parasites are also unlikely to effectively immunomodulate their hosts, so their ability to escape host immunity is weak. As used herein, the term "anti-molting" may refer to the effect of one or more pest control agents in preventing or delaying the molting process of pests. The molting process occurs when pests grow and shed their exoskeleton from one life stage to the next life stage, and is controlled by hormones and neurons, and the pest control agents herein may play one or more anti-molting effects on pests. As used herein, the terms "anti-fertility effect" and "anti-reproductive effect" are collectively referred to as "anti-fertility and anti-reproductive effects", and may include one or more effects on male or female reproduction. It is understood that the term "fertility" can refer to the actual number of offspring produced by a female or eggs released from a female, and the term "fecundity" can refer to the biological potential for reproduction, and because of their close relationship, the two terms as used herein can be used interchangeably unless otherwise indicated. Antifertility and anti-fertility effects can include reducing total gamete production in males and females, completely or partially inhibiting viable egg production, changing the anatomy and morphology of male or female gametes, changing the potential for fertilization of eggs, and reducing the total number of pregnant female pests.

[0069] In the application form, the amount of the pest control agent contained in the fish feed herein can be about 0.01 grams to 100 grams of pest control agent per kilogram of fish feed (g / kg), about 90 g / kg of fish feed, about 80 g / kg of fish feed, about 70 g / kg of fish feed, about 60 g / kg of fish feed, about 50 g / kg of fish feed, about 40 g / kg of fish feed, about 30 g / kg of fish feed, about 20 g / kg of fish feed, about 1 g to 10 g / kg of fish feed, about 2 g to 9 g / kg of fish feed, about 3 g to 7 g / kg of fish feed, about 4 g to 6 g / kg of fish feed, or about 5 g / kg of fish feed.

[0070] In various aspects, the fish feed herein can contain an amount of pest control agent and / or active ingredient effective to produce an inhibitory effect on one or more pests, including 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. / kg fish feed, 1.25g / kg fish feed, 1.5g / kg fish feed, 1.75g / kg fish feed, 2.0g / kg fish feed, 2.25g / kg fish feed, 2.5g / kg fish feed, 2.75g / kg fish feed, 3.0g / kg fish feed, 5.0g / kg fish feed, 5.25g / kg fish feed, 5.5g / kg fish feed, 5.75g / kg fish feed, 6.0g / kg fish feed, 6.25g / kg fish feed, 6.5g / kg fish feed, 6.75g / kg / kg fish feed, 7.0g / kg fish feed, 7.0g / kg fish feed, 7.25g / kg fish feed, 7.5g / kg fish feed, 7.75g / kg fish feed, 8.0g / kg fish feed, 8.25g / kg fish feed, 8.5g / kg fish feed, 8.75g / kg fish feed, 9.0g / kg fish feed, 9.25g / kg fish feed, 9.5g / kg fish feed, 9.75g / kg fish feed, 10.0g / kg fish feed, 15g / kg fish feed, 20g / kg or 100 g / kg of fish feed, or any amount within any of the foregoing concentration ranges.

[0071] It should be understood that the above concentrations correspond to an amount of about 0.001% to 10% by weight (% w / w) of the total pest control agent relative to the fish feed. In various aspects, the fish feed herein can contain an amount of a pest control agent effective to produce an inhibitory effect on one or more pests, including 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 any of the foregoing values. .

[0072] In various aspects herein, fish feeds may be administered to fish having a concentration of the pest control agent to fish feed selected from the group consisting of 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, or a range within any of the foregoing concentrations.

[0073] Pest control agents

[0074] Pest control agents suitable for use in fish feed and the pest control agent compositions herein can have an adverse effect on pests that feed on the host. It should be understood that when the host fish ingests the pest control agent as a component of its daily diet within a given duration, the pest control agent can be transferred to the body of the pest when the pest is fed from the host. Pest control agents can include any efficacy ingredients or active agents that affect, promote or contribute to eradicating or reducing pest infection or infestation of fish or fish schools. In addition, as a result of reducing, preventing or controlling infection or infestation, suitable pest control agents can alleviate or improve one or more symptoms associated with pest infection or infestation. Pest control agents used herein can have biological activity to one or more fish pests and one or more fish species.

[0075] It should be understood that when a fish ingests a pest control agent as described herein, the pest control agent is distributed systemically throughout the tissues and body fluids of the fish. Pests may be exposed to the pest control agent after ingesting the pest control agent through the skin, meat, blood, mucus, mucous membranes, or other tissues of the host organism. Regulation of pests (such as regulation of pest behavior and life cycle) occurs to reduce, prevent, or control infection or infestation of pests in fish. In various aspects, pests can be repelled or killed by the pest control agent herein. Therefore, the pest control agent herein can be provided to pests in fish feed or pest control agent compositions in an amount sufficient to regulate pest behavior.

[0076] Regulation of pests can have many effects on pest colonies, including ultimately reducing the number of live pests that can infect or infest host fish. Regulation of pests can include regulation of pest mortality. It should be understood that regulation of pest mortality can include reducing the number of live pests present on fish or in fish habitats. Regulation of pests can also include regulation of pest behavior, including changes in feeding habits, changes in feeding patterns, changes in appetite, changes in movement patterns, changes in swimming and migration patterns, changes in mating patterns, changes in development, changes in fertility, or any combination thereof compared to the pests found on control fish not fed with pest control agents. Changes in feeding patterns can include a reduction in feeding patterns. Changes in appetite can include a reduction in appetite. Changes in movement can include a reduction in movement. Changes in swimming and migration patterns can include a reduction in swimming and migration due to lethargy and lack of energy. Changes in mating patterns can include a reduction in mating patterns, which in turn can result in a reduction in the development or production of offspring. Alterations in development may include inhibition of development due to inhibition of the molting process resulting in a reduction in pest development or a delay in pest development. Alterations in fertility may include inhibition or delay in egg laying, failure to produce viable eggs, or a reduction in the total number of pregnant female pests.

[0077] In various aspects, the regulation of pests can include changes in the development of pests in their life cycle, including the regulation of growth or progress in a specific life stage, the regulation of growth or progress from one life stage to the next life stage (e.g., regulating molting), the regulation of laying eggs, the regulation of fertility, or any combination thereof. The regulation of growth or progress in a specific life stage can include stopping the growth of the organism and preventing further physical development, including the reduction of body size or sexual development. In some aspects, the regulation of growth or progress from one life stage to the next life stage can include preventing pests from transitioning from one life stage to the next life stage by suppressing the molting process. The regulation of laying eggs can include reducing female laying eggs, which can further cause the fertility of adult females to decrease. The regulation of fertility can include reducing the fertility of female and male pests.

[0078] In various aspects, applying a pest control agent to fish as described herein can also have a beneficial effect on the fish. The application of a pest control agent can impart a beneficial effect by improving the welfare of the fish by reducing the parasite load or total number of parasites in a given environment surrounding the fish. The application of a pest control agent can impart a beneficial effect by reducing the overall mortality rate within a fish population by alleviating or reducing the impact of parasitic infections or infestations on the fish population. The application of a pest control agent can impart a beneficial effect by minimizing or completely eliminating the impact on the quality and quantity of fish flesh within a fish population.

[0079] The fish feed described herein may contain or be supplemented with one or more pest control agents. When the fish feed contains at least two or more different pest control agents, each pest control agent may be individually active (or biologically active) and capable of regulating one or more of the behavior, development or fertility of the pest. Alternatively, the pest control agent may be a component of a pest control agent composition that can be fed to fish alone. Each pest control agent may be individually effective against one or more different pests as described herein.

[0080] Pest control agents suitable for use in the fish feed and pest control agent compositions herein may include one or more active agents, including synthetic or natural agents. The one or more synthetic or natural agents may include agents classified as active pharmaceutical ingredients, veterinary products, etc. In some aspects, active agents for the pest control agents herein may be obtained from plants belonging to the genus Azadirachta. Pest control agents may be obtained or extracted from Azadirachta indica (a tree commonly referred to as "Azadirachta indica"). Extracts prepared from plants belonging to the genus Azadirachta (e.g., Azadirachta indica) may include potent terpenoids, including one or more azadirachtinoid compounds. Azadirachtin compounds 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. Extracts from plants belonging to the genus Azadirachtin may also include many other components in varying amounts. In some aspects, the extract may include additional compounds such as the limonoid compounds salannin, nimbin, deacetyl salannin and 6-deacetyl azadirachtin. In various aspects, the extract may also include one or more azadirachtin compounds.

[0081] As used herein, the term "azadirachtin" may refer to a collective term applicable to a large group of active compounds, and is intended to cover not only all naturally occurring variants or derivatives of azadirachtin, including but not limited to azadirachtin A, B, D, E, F, G, H, I, K, but also all synthetic variants, fragments, analogs and derivatives thereof. In this regard, it should be understood that any azadirachtin variant, fragment, derivative or analog used herein should be functional in that they exhibit at least one inhibitory effect as described.

[0082] Azadirachtin can be obtained or extracted from any part of the neem plant (including, for example, leaves, stems, bark, fruits, seeds, or any combination thereof) by one or more extraction processes. Suitable extraction methods may include techniques utilizing mechanical pressing of neem seeds (i.e., kernels) and the use of non-polar solvents. Various solvent extraction techniques utilizing alcohol or water extraction processes, 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. Nos. 4,556,562, 5,695,763, and 11,096,404, the contents of which are incorporated herein by reference in their entirety.

[0083] For example, azadirachtin can be effectively recovered from the seeds of the neem tree. An exemplary method for recovering 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, the second extraction solvent comprising a non-aqueous solvent that is immiscible with water and has a higher solubility of azadirachtin than water or a surfactant having a turbidity temperature between 20°C and 80°C. Concentrated azadirachtin can be recovered from the second extraction solution and exhibits high activity as an insecticide and parasiticide. Extraction methods using polar solvents (e.g., water) produce extracts rich in polar components, such as azadirachtin compounds.

[0084] In various aspects, azadirachtin suitable for use herein includes azadirachtin A, whose scientific name is dimethyl[2aR-[2aα,3β,4β(1aR*,2S*,3aS*,6aS*,7S*,7aS*),4aβ,5α,7aS*,8β(E),10β,10aα,10bβ]]-10-(acetyloxy)octahydro-3,5-dihydroxy-4-methyl-8-[(2-methyl-1-oxo-2-butenyl)oxy]-4-(3a,6a,7,7a)-tetrahydro-6a-hydroxy-7a-methyl-2,7-methanofurano[2,3-b]epoxy[e]oxepin -1a(2H)-yl)-1H,7H-naphtho-[1,8-bc:4,4a-c']difuran-5,10a(8H)-dicarboxylate.

[0085] Azadirachtin A is the most abundant of a group of azadirachtin compounds. Azadirachtin A accounts for about 80% of the azadirachtin compounds in the neem seed kernel. The structural formula of azadirachtin A is:

[0086]

[0087] The pest control agent herein can include a neem extract, which is an aqueous extract. In various aspects, the neem extract can include an aqueous extract of neem seeds. The aqueous extract of neem seeds can include an aqueous extract of neem seed kernels. The aqueous extract of neem seed kernels can include an aqueous extract of whole neem seeds (including neem seed kernels and neem seed coatings). The aqueous extract of neem seeds can be in liquid form, or it can be dried to remove water to produce a powder form. For example, the neem extract herein can include an aqueous extract of neem seeds or an aqueous extract of neem seed kernels that have been dried into a powder.

[0088] It should be understood that the pest control agents described herein are not the same as neem extracts described as neem oil or solvent-first neem extracts. In various aspects, the pest control agents containing azadirachtin herein contain more abundant azadirachtin active ingredients, and in particular azadirachtin A, compared to neem oil and other oil-based formulations. This is because azadirachtin compounds (such as azadirachtin A) are relatively polar complex terpenoids with a large number of oxygen functional groups, which makes the molecule moderately water-soluble (e.g., a solubility of about 2 g / L). Therefore, compared to neem oil or solvent-first neem extracts, azadirachtin compounds (such as azadirachtin A) are present in extracts obtained using polar solvents at much higher concentrations. Without wishing to be bound by any particular theory, it is believed that the bioavailability of the active ingredients in the water-based extract of the extract enriched with azadirachtin A of the present disclosure to the target parasite can be greater than the bioavailability of the active ingredients in neem oil to the target parasite due to the increased solubility and miscibility of the water-based extract in water. Therefore, the pest control agent herein does not contain, consist of, or consist essentially of neem oil. The pest control agent of the fish feed provided herein may also contain, consist of, or consist essentially of azadirachtin A.

[0089] The pest control agent comprising a neem extract rich in azadirachtin A may include a pest control agent having at least 15% to 33% by weight of azadirachtin A. In various aspects, the pest control agent comprising a neem extract rich in azadirachtin A may include a pest control agent having at least 20% to 26% by weight of azadirachtin A. In various aspects, the pest control agent comprising a neem extract rich in azadirachtin A may include a pest control agent having at least 28% to 31% by weight of azadirachtin A. In some aspects, the pest control agent comprising a neem extract rich in azadirachtin A may include a pest control agent having at least 29% to 30% by weight of azadirachtin A. In other aspects, the pest control agent comprising a neem extract rich in azadirachtin A may include a pest control agent having at least 34% to 40% by weight of azadirachtin A. In various aspects, the pest control agent rich in azadirachtin A can include a pest control agent having 30±1% by weight of azadirachtin A. In various aspects, the pest control agent rich in azadirachtin A can include a pest control agent having 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, or 33%, or any amount falling within any of the foregoing ranges. In yet other aspects, the pest control agent comprising a neem extract rich in azadirachtin A can include a pest control agent having at least 34% to 45% by weight or at least 38% to 43% by weight of azadirachtin A. As used herein, unless otherwise specified, the terms "neem extract enriched with azadirachtin A" and "azadirachtin A enriched composition" are used interchangeably. Exemplary neem extract compositions suitable for use as azadirachtin A enriched pest control agent herein may include the formulations as summarized in Table 1.

[0090] Table 1. Exemplary Azadirachtin A-enriched pest control formulations

[0091]

[0092]

[0093] The pest control agent comprising the neem extract enriched with azadirachtin A may also contain other azadirachtin compounds in various concentrations. The azadirachtin compound may include an azadirachtin compound such as ≤19.0% w / w, or ≤6.0% w / w, or 4.0 to 6.0% w / w, or 5.6% w / w to 6.0% w / w of azadirachtin B; ≤13.0% w / w, or ≤5.0% w / w, or 2.5 to 5.0, or 4.0% w / w to 5.0% w / w of azadirachtin D; ≤5.0% w / w, or 1.0% w / w to 5.0% w / w, or 1.5% w / w to 2.0% w / w of azadirachtin E; ≤5.0% w / w, or 1.0% w / w to 5.0% w / w, or 1.5% w / w to 2.0% w / w of azadirachtin E; w / w of azadirachtin F; ≤5.0% w / w, or 1.0% w / w to 5.0% w / w, or 1.5% w / w to 2.0% w / w of azadirachtin G; ≤5.0% w / w, or 1.0% w / w to 5.0% w / w, or 2.5% w / w to 4.0% w / w of azadirachtin H; ≤5.0% w / w, or 1.0% w / w to 4.0% w / w, or 1.5% w / w to 2.5% w / w of azadirachtin I; and ≤5.0% w / w, or 1.0% w / w to 5.0% w / w, or 2.5% w / w to 4.0% w / w of azadirachtin K and / or other azadirachtin variants. The extract may also comprise ≤ 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 of azadirachtin.

[0094] Exemplary pest control agents suitable for use herein may include an aqueous extract of neem seeds that have been dried into a powder. The powder may include the appearance of a fine white powder. Exemplary pest control agents may include azadirachtin A at a concentration of 17% to 37% by weight, azadirachtin B at a concentration of 0% to 19% by weight, and azadirachtin D at a concentration of 0% to 13% by weight. Exemplary pest control agents may also include trace amounts of other limonoid compounds, including azadirachtin and saranlin.

[0095] Exemplary pest control agents comprising azadirachtin A-enriched compositions include, but are not limited to (Coromandel, Intl. Ltd., Telangana, India) or Technical (Coromandel International Ltd., Telangana, India) or any derivatives thereof.

[0096] Fish feed containing azadirachtin A

[0097] The fish feed herein may include a fish feed supplemented with the pest control agent azadirachtin A. The fish feed may be applied to various fish as part of a fish feed diet to control non-copepod parasitic pests in an aquaculture environment. The fish feed may be at least partially coated with a composition rich in azadirachtin A on the outer surface or completely coated with a composition rich in azadirachtin A on the outer surface. In some aspects, the fish feed herein may include a composition rich in azadirachtin A at least partially dispersed throughout the fish feed. In various aspects, a solid feed (such as a base feed particle) may also include a composition rich in azadirachtin A arranged on the surface or distributed throughout the fish feed, such as oil arranged in a porous matrix on the outer surface of the base feed particle. In various aspects, the fish feed herein may be at least partially coated with a composition rich in azadirachtin A on the outer surface, and may also have a composition rich in azadirachtin A at least partially dispersed throughout the fish feed. In various aspects, the fish feed may include one or more layers of a composition rich in azadirachtin A on the outer surface.

[0098] The fish feed herein may comprise a composition enriched with azadirachtin A at a concentration of about 0.01 to 100 grams per kilogram (g / kg) of fish feed, about 90 g / kg of fish feed, about 80 g / kg of fish feed, about 70 g / kg of fish feed, about 60 g / kg of fish feed, about 50 g / kg of fish feed, about 40 g / kg of fish feed, about 30 g / kg of fish feed, about 20 g / kg of fish feed, about 0.01 to 10 g / kg of fish feed, about 1 g to 10 g / kg of fish feed, about 2 g to 9 g / kg of fish feed, about 3 g to 7 g / kg of fish feed, about 4 g to 6 g / kg of fish feed, or about 5 g / kg of fish feed.

[0099] In various aspects, the fish feed herein may comprise a composition rich in azadirachtin A at a concentration of about 0.01 g of the composition rich in azadirachtin A per kilogram of fish feed (g / kg), 0.05 g / kg of fish feed, 0.1 g / kg of fish feed, 0.2 g / kg of fish feed, 0.3 g / kg of fish feed, 0.4 g / kg of fish feed, 0.5 g / kg of fish feed, 0.6 g / kg of fish feed, 0.7 g / kg of fish feed, 0.8 g / kg of fish feed, 0.9 g / kg of fish feed, 1.0 g / kg of fish feed. , 1.25g / kg fish feed, 1.5g / kg fish feed, 1.75g / kg fish feed, 2.0g / kg fish feed, 2.25g / kg fish feed, 2.5g / kg fish feed, 2.75g / kg fish feed, 3.0g / kg fish feed, 5.0g / kg fish feed, 5.25g / kg fish feed, 5.5g / kg fish feed, 5.75g / kg fish feed, 6.0g / kg fish feed, 6.25g / kg fish feed, 6.5g / kg fish feed, 6.75g / kg fish feed feed, 7.0g / kg fish feed, 7.0g / kg fish feed, 7.25g / kg fish feed, 7.5g / kg fish feed, 7.75g / kg fish feed, 8.0g / kg fish feed, 8.25g / kg fish feed, 8.5g / kg fish feed, 8.75g / kg fish feed, 9.0g / kg fish feed, 9.25g / kg fish feed, 9.5g / kg fish feed, 9.75g / kg fish feed, 10.0g / kg fish feed, 15g / kg fish feed, 20g / kg fish feed The present invention relates to a concentration of 1g / kg fish feed, 25g / kg fish feed, 30g / kg fish feed, 35g / kg fish feed, 40g / kg fish feed, 45g / kg fish feed, 50g / kg fish feed, 55g / kg fish feed, 60g / kg fish feed, 65g / kg fish feed, 70g / kg fish feed, 75g / kg fish feed, 80g / kg fish feed, 85g / kg fish feed, 90g / kg fish feed, 95g / kg fish feed, 100g / kg fish feed, or any amount within any of the foregoing concentration ranges.

[0100] It should be understood that the concentration of the above-mentioned azadirachtin A-rich composition is equivalent to about 0.001% w / w (weight percentage) to 10% w / w of the azadirachtin A-rich composition for fish feed. In various aspects, the fish feed herein may contain an azadirachtin A-rich composition, which is 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., the weight of the azadirachtin A-enriched composition to the weight of the fish feed), or any amount within any range of the foregoing values.

[0101] In various aspects herein, a fish feed having a composition enriched with azadirachtin A at a concentration selected from the group consisting of 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., the weight of the azadirachtin A-enriched composition to the weight of the fish feed), or a range within any of the foregoing concentrations.

[0102] Azadirachtins may be relatively unstable in water, however, when they are components of fish feed, such as when dispersed throughout or coated on fish feed, azadirachtins including azadirachtin A are at least temporarily stable, allowing them to exhibit their full biological activity during feeding. Any fish feed that falls to the seafloor will degrade after prolonged exposure to water. In addition, it should be noted that azadirachtin A or any neem extract agent disclosed herein presents minimal risk of toxic effects on fish or humans, and is therefore safe for use in wild and farmed fish. Arthropods and other invertebrates are sensitive to the active ingredient (i.e., azadirachtin A), while higher organisms including mammals are not affected. In addition, since azadirachtin A or any neem extract as described herein is readily soluble in water, they do not reside and accumulate in fish. In contrast, once application is stopped, the pest control agent may quickly lose its effectiveness because it is metabolized, degraded and / or excreted from the body. In the case of farmed fish stocks, this ensures that there is little to no harvest withdrawal period following administration of the fish feed or composition as described herein.

[0103] The fish feed provided herein can include a composition rich in azadirachtin A and one or more other agents. The one or more or other agents can include anti-ectoparasitic agents, antimicrobial agents (e.g., antibacterial agents, antifungal agents, antiviral agents), antiparasitic agents (e.g., anti-endoparasitic agents or anti-ectoparasitic agents) or anti-protozoal agents. The one or more or other agents can be mixed with the fish feed or coated on the fish feed, or layered in the fish feed. The one or more or other agents can be provided separately (e.g., in liquid or solid form) and can be applied separately (e.g., before or after the fish feed) or simultaneously with the fish feed (e.g., during the fish feed).

[0104] Immunomodulatory effects of dietary pest control agents

[0105] The pest control agent herein can play one or more immunomodulatory effects in the fish infested by the pests described herein. In various aspects, the pest control agent herein can play an immunomodulatory effect by alleviating or eliminating any immune system disorder experienced by the fish. The immune system disorder caused by the parasite may include suppressing protective, beneficial immune responses while aggravating non-protective or harmful immune responses. In various aspects, the use of the pest control agent herein can play an immunomodulatory effect by reducing, controlling or preventing the parasite load on the animal, wherein the parasite load may include the number of parasites present during infection or infestation. By reducing the parasite load, the pest control agent can reduce the undesirable inflammatory response that is not controlled by the immune system in response to infection or infestation, or can stimulate protective immune protection and physiological responses. If not controlled, the overactivated immune system can cause various types of damage to the tissues of the host fish. Therefore, in some aspects, the reduction of undesirable inflammatory responses can be an indirect effect due to the reduction of the number of parasites on the fish. Similarly, the stimulation of protective immune responses and physiological responses can also be the result of the direct or indirect effects of the pest control agent on parasites or hosts.

[0106] In various aspects, the reduction of undesirable inflammatory response can be a direct response to the pest control agent itself. In other aspects, the pest control agent can play an immunomodulatory role in the following ways: reduce, control or prevent the infection or infestation of pests, so that there is no inhibition of protective, beneficial immune response, and there is no upregulation of non-protective or harmful immune response, thereby allowing the immune system to work optimally. It should be understood that a beneficial protective immune response can include the activation of the immune system to parasitic organisms, protective immune responses and protective physiological responses, the repair of damage caused by non-protective immune responses, and subsequent parasite removal. Harmful non-protective immune responses can include responses that can cause immunopathology (such as tissue damage caused by immune response disorders). Harmful non-protective immune responses can also include responses that negatively affect the health of fish by stopping, preventing protective immune responses, or reducing the effectiveness of protective immune responses when fish face immune challenges (such as overactivation of regulatory and anti-inflammatory pathways or activation of competitive, non-protective immune responses induced by parasites). In various aspects, the immunomodulatory effects exerted by the pest control agent can include reducing, controlling or preventing infection or infestation, thereby minimizing the negative effects of the infection or infestation, including minimizing excess mortality, minimizing deleterious effects on meat quality, and minimizing reductions in animal welfare, and allowing fish to combat other pathogens and stressors, including heat stress, hypoxic stress, oxidative stress, etc., compared to infected or infested fish to which the pest control agent is not applied.

[0107] Method for preparing fish feed

[0108] The disclosure herein provides a method for preparing a fish feed comprising one or more pest control agents, such as a neem extract comprising a composition rich in azadirachtin A. The method may include the steps of providing a base feed and applying an amount of a pest control agent to the surface of the base feed. The base feed may be formed from various raw materials as described elsewhere herein. For example, the method may include coating the base feed with an amount of a pest control agent. The base feed may be at least partially coated with a pest control agent on the outer surface, or completely coated with a pest control agent on the outer surface. In some aspects, the base feed herein may include a pest control agent at least partially dispersed throughout the base feed. In various aspects, the base feed herein may be at least partially coated with a pest control agent, and may also have a pest control agent at least partially dispersed throughout the base feed. In some aspects, the pest control agent composition may be distributed throughout the base feed (such as throughout a porous matrix of the base feed). In various aspects, the base feed may be coated with more than one layer of a pest control agent on the outer surface, wherein each layer may contain the same pest control agent, or in some aspects, each layer may contain different pest control agents.

[0109] The pest control agent can be incorporated into or mixed into the base feed by various methods. In various aspects, the base feed can be prepared using an extrusion process or a pressing process. The pest control agent can be mixed with the matrix during its manufacture so that it is distributed in all or part of the fish feed. Once the pest control agent is mixed with the base feed, the base feed and the pest control agent mixture can be formed into, for example, granules, flakes, tablets, powders, or any desired form. In the case of temperature-sensitive pest control agents, such pest control agents can be added to the base feed after the base feed has been formed into one of the various forms shown. In various aspects, the pest control agent can be sprayed onto the base feed that has been formed into granules, flakes, tablets, etc. For example, the pest control agent can be applied to the base feed as one or more layers or top coatings. In various aspects, the pest control agent can be applied to the outer surface of the granules or flakes-in this way, the fish feed granules or flakes can become completely or partially coated with the pest control agent. One or more agent layers or coatings can be applied to the outer surface of the fish feed flakes or granules. Any agent layer or coating can be "sealed" or protected by applying one or more additional sealing material coatings or layers. In various aspects, the pest control agents herein may be dispersed in one or more oils or fractions thereof and may be incorporated into a porous matrix within a fish feed by a vacuum coating process.

[0110] For example, the agent layer or coating can be sealed by applying a layer or coating of oil (such as fish oil). In various aspects, one or more additional fish feed layers or coatings can be applied to the (optionally sealed) fish feed coating or layer. In this way, any given fish feed flakes or particles can contain multiple fish feeds, sealing material layers and / or pest control agent layers. It should be understood that the fish feed described herein can be at least partially coated with a pest control agent on the outer surface. In various aspects, the fish feed herein can include a pest control agent that is at least partially dispersed throughout the fish feed. In various aspects, the fish feed herein can include a pest control agent that is at least partially coated with a pest control agent and is at least partially dispersed throughout the fish feed.

[0111] Methods for incorporating a pest control agent into a fish feed may include incorporating the pest control agent so that the final concentration of the pest control agent in the fish feed includes about 0.01 grams of pest control agent per kilogram of fish feed (g / kg) to about 1000 g / kg, or 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, 20 g / kg, 30 g / kg, 40 g / 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 any of the foregoing ranges. Concentrations are described in more detail herein with reference to fish feed.

[0112] Methods for incorporating a pest control agent into fish feed may include determining the final concentration of the pest control agent incorporated as part of the fish feed. Determination of the final concentration may include sampling the fish feed using various quantitative analytical methods. For example, the fish feed sample may be extracted by overnight protein precipitation in methanol. After extraction, the sample may be analyzed using Supel TM The samples were cleaned with QuE Z-Sep+ (Sigma Aldrich, St. Louis, Missouri, USA) adsorbent, which is a silica-based material with an activated zirconium oxide phase, a particle size of approximately 50 μm, and a pore size of 70 Å. The resulting extraction solution may be filtered through a polytetrafluoroethylene filter having a pore size of 0.2 μm or greater. Analysis of the final concentration of the pest control agent in the fish feed sample may be performed using high performance liquid chromatography with ultraviolet detection (HPLC-UV).

[0113] The methods herein may include extracting a neem extract enriched in azadirachtin A, including extracts having at least 15 wt% to 33 wt% azadirachtin A, at least 28 wt% to 31 wt% azadirachtin A, and at least 29 wt% to 30 wt% azadirachtin A.

[0114] The method may further include the step of sealing the azadirachtin A-rich composition applied to the surface of the fish feed. The azadirachtin A-rich composition may be sealed by applying a coating of fish oil to the azadirachtin A-rich composition-coated fish feed. Any sealing substance for sealing the azadirachtin A-rich composition may be applied so that it coats all or part of the azadirachtin A-rich composition coating.

[0115] Pest control composition

[0116] The present disclosure also provides a pest control agent composition for application to fish, wherein the pest control agent composition may include one or more pest control agents. It should be understood that the pest control agent composition is not a fish feed, but is intended to be applied to fish separately or in addition to fish feed. The pest control agent composition can be provided separately for application before, during or after the application of fish feed. Therefore, in various aspects, the pest control agent composition herein may be suitable for use as a veterinary product or dietary supplement for reducing, preventing or controlling pest infections or infestations in fish in some aspects. The pest control agent composition may be applied to fish at a concentration described elsewhere herein. For example, the pest control agent composition herein may be applied in an amount of about 0.01 grams of pest control agent / kilogram of fish feed (g / kg) to about 100g / kg, as described elsewhere herein. In various aspects, the pest control agent composition comprises a neem extract rich in azadirachtin A.

[0117] The pest control agent composition herein may include an azadirachtin extract rich in azadirachtin A. The pest control agent composition may include a liquid, solid or semisolid form, and may also include one or more of an excipient, a diluent, a carrier, a vitamin, a mineral or a combination thereof. The pest control agent composition may be in the form of a dietary supplement provided as any one of granules, flakes, pellets, powders, tablets, pills, capsules, etc. In various aspects, the pest control agent composition herein may be formed into many shapes and sizes. In various aspects, the fish feed herein may be in the shape of a triangle, square, rectangle, sphere, rhombus, cylinder, spherical, clover-shaped, amorphous, etc. The fish feed may be formed by a method including one or more of extrusion, distillation, cold pressing, high pressure processing, etc.

[0118] Alternatively, the pest control agent composition can be provided in a form that can be eaten by fish but does not provide nutrition to the fish. For example, the pest control agent composition can include a veterinary product that can contain a substance or combination of substances for managing or preventing fish diseases. The pest control agent composition can also be formulated for parenteral administration. Therefore, the pest control agent composition can include a pharmaceutically acceptable carrier, diluent or excipient or a combination thereof. In addition, the pest control agent composition can be sterile.

[0119] The pest control agent composition herein can be included in one or more types of fish feeds intended to be mixed 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 surface feed, a liquid drenching agent, or a combination thereof.

[0120] The pest control agent in the pest control agent composition herein may include one or more agents for reducing, preventing or controlling infections or infestations caused or contributed to by one or more pests (including any type of worms, worms, trematodes, bacteria, viruses, fungi and protozoa, as described elsewhere). Each pest control agent contained in the pest control agent composition may be individually capable of reducing, preventing or controlling one or more of parasitic, bacterial, viral, fungal or protozoan infections or infestations. For example, the pest control agent composition herein may include a pest control agent composition that exhibits one or more inhibitory effects, including antiparasitic effects, antibacterial effects, antiviral effects, antifungal effects, antiprotozoal effects, or any combination thereof.

[0121] The pest control agent composition can be applied before, during or after applying any fish feed. In some respects, the pest control agent composition can be applied together with the fish feed that does not contain the pest control agent. In some respects, the pest control agent composition can be co-applied with the fish feed containing the pest control agent. When co-applied with the fish feed containing the pest control agent, the independent pest control agent composition can include the pest control agent identical with the pest control agent in the fish feed, or it can be the pest control agent different from the pest control agent in the fish feed. When co-applied with the fish feed containing the pest control agent, the concentration of the independent pest control agent composition can be identical with the concentration of the pest control agent in the fish feed, or it can be different from the concentration of the pest control agent in the fish feed. The pest control agent composition herein can be included in the fish feed in the form of a veterinary product or any complete and balanced dietary supplement for fish feed, or can be provided as a component of a complete fish feed.

[0122] Method of applying pest control agents to fish

[0123] The pest control agents herein can be applied to fish in fish feed and pest control agent compositions. The management method utilizing the pest control agent in fish feed can be referred to as an in-feed agent delivery method. Therefore, the present disclosure provides an in-feed agent delivery method for reducing, preventing or controlling pests. It should be understood that the in-feed agent delivery method is not a method for topically applying the pest control agent to the target pest. The management method herein can also utilize a non-feed form of the pest control agent, such as a veterinary product or a dietary supplement. Therefore, the present disclosure also provides a veterinary product or a dietary supplement as a medicament for reducing, preventing or controlling pests.

[0124] Pest control agents not included in fish feed can be applied to fish as supplements to fish feed in separate pest control agent compositions, such as in the form of veterinary products or dietary supplements. Fish feed can be applied simultaneously or separately with the pest control agent composition. It should be noted that various pest control agents herein can be applied to sick fish, fish infested with parasites, otherwise healthy fish (to prevent parasitic infection), or fish with weaker aggressiveness due to different infections or conditions unrelated to pest infection or infestation. It should be understood that sick or less aggressive fish may eat less, and therefore may ingest lower concentrations of pest control agents. Therefore, management methods based on the use of both fish feed and pest control agent compositions may be particularly useful for managing fish whose appetite is affected by disease, infection, infestation (or fish with weaker aggressiveness that usually eat less fish feed). In addition, for fish with weaker aggressiveness or low food intake, the use of pest control agent compositions and fish feed supplemented with pest control agents simultaneously can promote or ensure the correct concentration of applying pest control agents to fish.

[0125] The concentration of the pest control agent added to the fish feed or pest control composition herein can be an amount effective to achieve the desired regulation of the behavior, development or mortality of the pest as discussed elsewhere herein. It should be understood that the exact amount of the pest control agent to be added to the fish feed or pest control composition herein can vary depending on, for example, the species of fish, the number of fish to be fed, the degree of infection or infestation, etc. Other factors that affect the amount of the pest control agent added to the fish feed or pest control composition include, for example, the presence of possible competitors for the feed (i.e., other non-target animal species that may eat the fish feed), the type of pest to be controlled, the age and maturity of the pest, the age and maturity of the fish, the season, the type of water (e.g., pH, salinity, purity, temperature), and the aggressiveness of the fish. It should be understood that the concentration of the pest control agent added to the fish feed or pest control composition herein can include an amount effective to achieve the desired effect of regulating the behavior and development of the pest, wherein the effective amount includes one or more concentrations or concentration ranges as described herein. It will be appreciated that an effective amount may be determined by comparison to a control fish or fish population not fed the pest control agent.

[0126] The fish feed and the pest control agent composition can be formulated so that the concentration of the pest control agent applied to the fish via the fish feed or the pest control agent composition can be about 0.01 mg to 100 mg of the pest control agent / kg body weight / day (mg / kg / day), 1 mg to 90 mg / kg / day, 1 mg to 80 mg / kg / day, 1 mg to 70 mg / kg / day, 1 mg to 60 mg / kg / day, 5 mg to 50 mg / kg / day, 10 mg to 40 mg / kg / day, 15 mg to 35 mg / kg / day, 20 mg to 30 mg / kg / day, 0.01 mg to 10 mg / kg / day, or 0.01 mg to 5.0 mg / kg / day. In various aspects, the pest control agent is applied to the fish in the fish feed at a target concentration of 0.01 mg to 5.0 mg of azadirachtin A / kg body weight / day. In various aspects, the pest control agent is applied to fish in fish feed at a target concentration of 1.5 mg to 2.5 mg azadirachtin A / kg body weight / day. In some aspects, the pest control agent is applied to fish in fish feed at a target concentration of 2.6 mg to 5.0 mg azadirachtin A / kg body weight / day.

[0127] It should be understood that the amount of pest control agent applied to fish can include an amount effective to produce an inhibitory effect on one or more pests, which is in the range of about 0.01 mg to 100 mg / kg / day (e.g., mg pest control agent / kg body weight / day), including 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, 10 ... .09mg / kg / day, 0.10mg / kg / day, 0.20mg / kg / day, 0.30mg / kg / day, 0.40mg / kg / day, 0.50mg / kg / day, 0.60mg / kg / day, 0.70mg / kg / day, 0.80mg / kg / day, 0.90mg / kg / day, 1.0mg / kg / day, 2.0mg / kg / day, 3.0mg / kg / day, 4.0mg / kg / day, 5.0mg / kg / day, 6.0mg / kg / day, 7.0mg / kg / day .0mg / kg / day, 8.0mg / kg / day, 9.0mg / kg / day, 10.0mg / kg / day, 11.0mg / kg / day, 12.0mg / kg / day, 13.0mg / kg / day, 14.0mg / kg / day, 15.0mg / kg / day, 16.0mg / kg / day, 17.0mg / kg / day, 18.0mg / kg / day, 19.0mg / kg / day, 20.0mg / kg / day, 25.0mg / kg / day, 30.0mg / kg / day In some embodiments, the present invention relates to an inhibitory effect of at least one embodiment of the present invention. The inhibitory effect of at least one embodiment of the present invention may be at least one embodiment of the present invention. The inhibitory effect of at least one embodiment of the present invention may be at least one embodiment of the present invention. The inhibitory effect of at least one embodiment of the present invention may be at least one embodiment of the present invention.

[0128] Fish feed or pest control agent composition can be applied for a period of time, as long as the desired inhibitory effect is reached. For example, the pest control agent composition or fish feed can be applied in about 10 to 20 days or about 14 to 18 days. It should be understood that the pest control agent composition, fish feed or both can be applied continuously 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 any number of days falling within any of the aforementioned ranges. In various fields, the pest control agent composition or fish feed herein can be applied for a longer period of time, such as more than 20 days. In various fields, fish feed or pest control agent can be applied for at least 11 days. In various fields, fish feed or pest control agent can be applied for at least 14 days. It should be understood that the time required for applying the pest control agent composition or fish feed herein can have a variable length, so as to target the developmental life stage of the pests present in the school of fish, for water temperature, pest control agent concentration or any combination thereof. In some aspects, the pest control agents herein can be applied prophylactically in the diet of fish in an amount effective to prevent pest infection or infestation from occurring within the fish population. It should also be understood that the pest control agents herein can be applied prophylactically in the diet at any time during the life cycle of the fish, such as from stocking to harvest, seasonally, or during an outbreak of infection or infestation within a population or a nearby farm.

[0129] In various management methods, the pest control agent of this paper can be applied for several days non-continuously, wherein the pest control agent is applied after a period of rest in the predetermined time period, and then again after a period of rest in the predetermined time period, and so on. For example, in some aspects, the pest control agent can be applied for three days every 10 days. In other aspects, the pest control agent can be applied for seven days every 14 days. The method of applying the pest control agent and then resting for a period of time within the predetermined time can be reused as required, or until reducing, preventing or controlling pests to infect or infest. It should be understood that the pest control agent can be applied for a predetermined time period, including applying 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days every 5 days to 30 days of rest between application.

[0130] During application, the pest control agent composition or fish feed can be applied multiple times a day as needed to achieve an inhibitory effect. For example, the pest control agent composition or fish feed described herein can be applied about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times a day.

[0131] The use of fish feed and pest control agent compositions to apply the pest control agent herein to fish can be performed by various methods. In one aspect, a method for reducing, preventing or controlling non-copepod parasite infections or infestations in fish schools. The method may include providing a fish feed comprising a neem extract rich in azadirachtin A, the neem extract comprising 15% to 33% by weight of azadirachtin A. The method may include applying a fish feed comprising a neem extract rich in azadirachtin A to one or more fish, wherein the fish feed provides a concentration of 0.01 mg to 5.0 mg azadirachtin A / kg body weight / day to the one or more fish.

[0132] In one aspect, a method for reducing, preventing or controlling a non-copepod parasite infection or infestation in fish is provided. The method may include providing a pest control agent composition comprising a pest control agent comprising a neem extract enriched in azadirachtin A. The method may include applying the pest control agent composition to one or more fish during an infection or infestation for 1 to 20 days, wherein the concentration of azadirachtin A applied to the fish by the pest control agent composition is about 0.01 mg to 5 mg azadirachtin A / kg body weight / day.

[0133] For the methods herein, any of the methods may involve administering a fish feed to a fish or fish population experiencing a non-copepod parasite infection or infestation caused by one or more organisms including Giardia ovale, Centrocariasis, Cryptocaryon irritans, Species of the genus Leptospira, Trichodina, Myxosporea salmoninae, Ichthyophthirius multifida, Kudaoa scombrida, Iodine myxomycetes, Neoparamoebae, Saprolegnia parasiticus, Monogenea, Trichodina spp., or Arogam leech.

[0134] For the methods herein, any method can include wherein the concentration of azadirachtin A comprises a concentration in an amount effective to increase the efficacy of azadirachtin A against non-copepod parasitic infection or infestation compared to fish fed a diet lacking the neem extract enriched in azadirachtin A.

[0135] For the methods herein, any method may comprise administering the fish feed to the farmed fish for at least 14 days.

[0136] For the methods herein, any method may include wherein the neem extract enriched in azadirachtin A is administered to the fish at a concentration of 1.5 mg to 2.5 mg azadirachtin A / kg body weight / day.

[0137] For the methods herein, any method may include wherein the neem extract enriched in azadirachtin A is administered to the fish at a concentration of 2.6 mg to 5.0 mg azadirachtin A / kg body weight / day.

[0138] For the methods herein, any method can include wherein the fish feed further comprises one or more components including an antibacterial, antifungal, antiviral, antiparasitic, or antiprotozoal agent.

[0139] For the methods herein, any method may include wherein the fish feed is administered to a fish species belonging to one or more families including: Cyprinidae, Cichlidae, Panfish, Sciaenidae, Serranidae, Carpidae, Sparidae, Perchidae, Lycaenidae, Mugilidae, Cypriniformes, Perchidae, Channaidae, Tilapia, and Salmonidae.

[0140] For the methods herein, any method may include wherein the pest control agent is configured to produce an inhibitory effect including one or more of an antiparasitic effect, an antibacterial effect, an antiviral effect, an antifungal effect, or an antiprotozoal effect.

[0141] For the methods herein, any method may include wherein the neem extract enriched in azadirachtin A does not comprise neem oil.

[0142] For the methods herein, any method can include wherein the pest control agent is provided to the pest in the fish feed in an amount sufficient to modify the behavior of the pest.

[0143] For the methods herein, any method can include wherein modulating the behavior of the pest includes one or more of: a change in feeding habits, a change in feeding patterns, a change in appetite, a change in movement patterns, or a change in mating patterns compared to the pest found on a control animal not fed the pest control agent.

[0144] For the methods herein, any method may include wherein a neem extract enriched 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 solvent comprising a non-aqueous solvent that is immiscible with water and has a higher solubility for azadirachtin than water or a surfactant having a turbidity temperature between 20°C and 80°C; and recovering concentrated azadirachtin from the second extraction solution.

[0145] Targeted management approach for pest control application

[0146] The method for applying the pest control agent herein can include a targeted management method, which utilizes a targeted concentration and a targeted exposure duration to maximize the antiparasitic effect of the pest control agent on each life stage of the pest. The targeted management method can include applying the pest control agent by one or more fish feeds or pest control agent compositions as described elsewhere herein. The targeted concentration can include an amount that effectively produces an inhibitory effect on one or more pests, wherein the inhibitory effect can include one or more of an anti-feeding effect, an anti-molting effect, an anti-fertility effect, and an anti-fertility effect or an anti-parasitic effect. The targeted exposure duration can include the time period required for achieving the desired inhibitory effect. It should be understood that in various aspects, some life stages will require a smaller targeted concentration and a targeted exposure duration, while other life stages will require a larger targeted concentration and a longer targeted exposure duration.

[0147] By targeting different life stages with concentration and exposure duration, management methods can be customized to maximize efficacy at the lowest concentration of the pest control agent required for the inhibition. By utilizing the pest control agent at the lowest concentration as much as possible, management methods can achieve high efficacy at lower targeted concentrations, so as to keep the management costs of farmers lower. As described elsewhere herein, pest control agents can include those neem extracts rich in azadirachtin A, which can produce inhibitions for one or more pests, including one or more effects of concentration and spread for reducing, preventing or controlling various parasites. As mentioned above, azadirachtin A has an antifeedant property for pests, wherein the antifeedant property can play one or more antifeedant effects, including malnutrition, stunted development, molting obstruction or delay and death, all of which can contribute to the excess mortality of pests over time at all life stages.

[0148] The inhibitory effect may include a reduction in the total number of pests molting from one developmental life stage to another developmental life stage compared to fish infected or infested with one or more pests fed a diet lacking a neem extract rich in azadirachtin A. The inhibitory effect may also include a reduction in the total number of adult females or a reduction in the total number of adult males in a pest population exposed to fish fed a diet containing azadirachtin A compared to fish infected or infested with one or more pests fed a diet lacking a neem extract rich in azadirachtin A.

[0149] The inhibitory effect of the pest control agent can lead to excess mortality in the pest population. For example, pest mortality can increase with each day that the pest is exposed to the target concentration during the targeted exposure duration, and is referred to herein as "extra daily mortality". Pest mortality also increases at each molting stage, and is referred to herein as "extra mortality during molting". In addition to the extra daily mortality due to malnutrition, a neem extract rich in azadirachtin A can substantially inhibit or prevent pests from developing from one stage to the next, thereby resulting in extra mortality during molting. Further, a neem extract rich in azadirachtin A can exhibit an ovicidal effect on female pests, wherein the ovicidal effect can include reduced egg laying, reduced egg vitality, reduced egg size, and reduced hatching time, thereby further resulting in extra mortality during molting.

[0150] The targeted management methods disclosed herein can be customized to expose pests to pest control agents within one or more developmental windows to target various or multiple life stages. It should be understood that the developmental life stage composition of the pest population present during infection or infestation of farmed fish varies in response to various environmental factors, including season, ocean currents, water temperature, or the use of physical lice control systems. The targeted management methods herein may include alternating between applying pest control agents with a lower targeted concentration range and a higher targeted concentration range according to the life stage present at a given time. Further, the methods herein may include applying pest control agents at the beginning of an infection or infestation, midway through an infection or infestation, or in the later stages of an infection or infestation, or a combination thereof. It should be understood that the beginning of an infection or infestation may include the 0th to the 9th day after the infection of the pest; the midway through an infection or infestation may include the 10th to the 19th day after the infection of the pest; and the later stages of an infection or infestation may include the 20th or more days after the infection of the pest.

[0151] For example, some methods may include alternating between applying a targeted concentration of 0.01 mg / kg fish / day to 2.5 mg / kg fish / day for 14 days to target the early life stages of the pest; and then applying a targeted concentration of 2.6 mg / kg fish / day to 5.0 mg / kg fish / day for 14 days to target the later life stages of the pest.

[0152] In some aspects of the methods herein, a second management method including exposure to a pest control agent may be performed immediately after a first management method including exposure to a pest control agent, while in other aspects, a second management method including exposure to a pest control agent may be performed after waiting for a predetermined period of time after a first management method including exposure to a pest control agent. The predetermined period of time may include 1 day to 60 days, or any number of days falling within the range of 1 day to 60 days. In various aspects, the predetermined period of time may include more than 60 days. It should be understood that during the predetermined period of time, the fish exposed to the pest control agent may be allowed to rest and recover from any external stress imposed on it during the first or second management method. In some aspects, the methods herein may include repeating a cycle of first pest control agent exposure and second pest control agent exposure, as long as it is necessary to reduce or prevent infection or infestation of pests described herein. In various aspects, a third pest control agent exposure different from the first or second pest control agent exposure may be used, wherein the third pest control agent exposure includes a pest control agent concentration different from either the first management method or the second management method.

[0153] It should be understood that the definitions described herein apply to all aspects described unless otherwise stated.

[0154] 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 non-exclusive "or" unless otherwise indicated. All publications, patents, and patent documents cited in this document are incorporated herein by reference in their entirety, as if individually incorporated by reference. In the event of an inconsistency between the usages of this document and those documents so incorporated by reference, the usage in the incorporated references should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0155] Values ​​expressed in range format should be interpreted in a flexible manner to include not only the values ​​explicitly listed as the limits of the range, but also all individual values ​​or subranges covered within the range, as if each value and subrange were explicitly listed. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also individual values ​​(e.g., 1%, 2%, 3% and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the specified range. Unless otherwise indicated, the statement "about X to Y" has the same meaning as "about X to about Y". Similarly, unless otherwise indicated, the statement "about X, Y or about Z" has the same meaning as "about X, about Y or about Z".

[0156] Unless expressly stated, ppm (parts per million), percentages and ratios are based on weight. Percentages based on weight (%w / w or w / w%) are also referred to herein as weight percent (wt%) or weight percent (%wt.).

[0157] Exemplary embodiments of the present invention are as follows;

[0158] Embodiment 1: A method for reducing, preventing or controlling a non-copepod parasite infection or infestation in a fish population, the method comprising:

[0159] Provided is a fish feed comprising a neem extract rich in azadirachtin A, wherein the neem extract

[0160] The extract contains 15 wt % to 33 wt % of azadirachtin A;

[0161] administering the fish feed comprising the neem extract enriched in azadirachtin A to one or more fish; and

[0162] wherein the fish feed provides a concentration of 0.01 mg to 5.0 mg azadirachtin A / kg body weight / day to the one or more fish.

[0163] Embodiment 2: A method according to embodiment 1, wherein the non-copepod parasitic infection or infestation is caused by one or more parasitic organisms, and the one or more parasitic organisms include Giardia ovale, Centrocariasis, Cryptocaryon irritans, Species of the genus Leptospira, Trichodina, Myxosporea salmoninae, Ichthyophthirius multifida, Kudaoa serpentinae, Iodine bubble worm, Neoparamoebae, Saprolegnia parasiticus, Monogenea, Trichodina spp., or Arugam ceylon leech.

[0164] Embodiment 3: The method according to embodiment 1, wherein the fish feed is administered to farmed fish for at least 11 days.

[0165] Embodiment 4: The method according to embodiment 1, wherein the fish feed is administered to farmed fish for at least 14 days.

[0166] Embodiment 5: The method of embodiment 1, wherein the concentration of azadirachtin A comprises a concentration in an amount effective to increase the efficacy of azadirachtin A against non-copepod parasitic infection or infestation compared to fish fed a diet lacking the azadirachtin A-enriched neem extract.

[0167] Embodiment 6: The method according to any one of embodiments 1-5, wherein the neem extract enriched in azadirachtin A is administered to the fish at a concentration of 1.5 mg to 2.5 mg azadirachtin A / kg body weight / day.

[0168] Embodiment 7: The method according to any one of embodiments 1-6, wherein the neem extract enriched in azadirachtin A is administered to the fish at a concentration of 2.6 mg to 5.0 mg azadirachtin A / kg body weight / day.

[0169] Embodiment 8: The method of any one of Embodiments 1-7, wherein the fish feed further comprises one or more components comprising an antibacterial, antifungal, antiviral, antiparasitic, or antiprotozoal agent.

[0170] Embodiment 9: The method according to any one of embodiments 1-8, wherein the fish feed is administered to fish species belonging to one or more families including: Cyprinidae, Cichlidae, Panfish, Sciaenidae, Serranidae, Carangidae, Sparidae, Perchidae, Lycoperdontidae, Mugilidae, Cypriniformes, Percidae, Channaidae, Tilapia and Salmonidae.

[0171] Embodiment 10: The method according to any one of Embodiments 1-9, wherein the pest control agent is configured to produce an inhibitory effect, the inhibitory effect comprising one or more of an antiparasitic effect, an antibacterial effect, an antiviral effect, an antifungal effect, or an antiprotozoal effect.

[0172] Embodiment 11: The method according to any one of Embodiments 1-10, wherein the neem extract enriched in azadirachtin A does not comprise neem oil.

[0173] Embodiment 12: The method of any one of Embodiments 1-11, wherein the pest control agent is provided to the pest in the fish feed in an amount sufficient to modulate the behavior of the pest.

[0174] Embodiment 13: The method of any one of Embodiments 1-12, wherein modulating the behavior of the pest comprises one or more of a change in feeding habits, a change in feeding patterns, a change in appetite, a change in movement patterns, or a change in mating patterns, as compared to pests found on control animals not fed the pest control agent.

[0175] Embodiment 14: The method of embodiment 12, wherein the change in eating pattern comprises a decrease in eating pattern.

[0176] Embodiment 15: The method of embodiment 12, wherein the change in appetite comprises a decrease in appetite.

[0177] Embodiment 16: A method according to embodiment 12, wherein the change in movement pattern includes a reduction in movement.

[0178] Embodiment 17: The method of embodiment 12, wherein the change in mating pattern comprises a decrease in mating pattern.

[0179] Embodiment 18: The method of Embodiment 17, wherein the alteration in mating pattern comprises a reduction in the number of live pests present on the fish or in the fish's habitat as compared to pests found on control fish not fed the pest control agent.

[0180] Embodiment 19: The method according to any one of embodiments 1-18, wherein the neem extract enriched in azadirachtin A is obtained by a method comprising the steps of:

[0181] Neem seeds are available;

[0182] crushing the neem seeds;

[0183] Extract azadirachtin from the crushed seeds using water;

[0184] adding a second extraction solution, the second extraction solution comprising:

[0185] a non-aqueous solvent that is immiscible with water and has a higher solubility for azadirachtin than water; or

[0186] a surfactant having a turbidity temperature between 20°C and 80°C; and

[0187] Concentrated azadirachtin is recovered from the second extraction solution.

[0188] Embodiment 20: A method for reducing, preventing or controlling a non-copepod parasite infection or infestation in fish, the method comprising:

[0189] Provided is a pest control agent composition comprising a pest control agent, wherein the pest control agent comprises a neem extract rich in azadirachtin A; and

[0190] applying the pest control composition to one or more fish during an infection or infestation period for a period of 1 to 20 days;

[0191] The concentration of azadirachtin A applied to the fish via the pest control agent composition is about 0.01 mg to 5 mg azadirachtin A / kg body weight / day.

Claims

1. A method for reducing, preventing or controlling a non-copepod parasite infection or infestation in a fish population, the method comprising: include: Providing a fish feed comprising a neem extract enriched with azadirachtin A, wherein the neem extract comprises 15 wt % to 33 wt % of azadirachtin A; administering the fish feed comprising the neem extract enriched in azadirachtin A to one or more fish; and wherein the fish feed provides a concentration of 0.01 mg to 5.0 mg azadirachtin A / kg body weight / day to the one or more fish.

2. The method of claim 1, wherein the non-copepod parasitic infection or infestation is caused by one or more parasitic organisms, the one or more parasitic organisms comprising Giardia ovale, Centrocera, Cryptocaryon irritans, Species of the genus Leptocera, Trichodina, Myxosporea salmoninae, Ichthyophthirius multifida, Kudaoa serpentinae, Iodine myxoma, Neoparamoebae, Saprolegnia parasiticus, Monogenea, Trichodina spp., or Leech of Arugam.

3. The method according to claim 1 or 2, wherein the fish feed is administered to farmed fish for at least 11 days.

4. The method of any one of claims 1-3, wherein the concentration of azadirachtin A comprises a concentration in an amount effective to increase the efficacy of azadirachtin A against non-copepod parasite infection or infestation compared to fish fed a diet lacking the neem extract enriched in azadirachtin A.

5. The method according to any one of claims 1 to 4, wherein the neem extract enriched in azadirachtin A is administered to the fish at a concentration of 1.5 mg to 2.5 mg azadirachtin A / kg body weight / day.

6. The method according to any one of claims 1 to 5, wherein the neem extract enriched in azadirachtin A is administered to the fish at a concentration of 2.6 mg to 5.0 mg azadirachtin A / kg body weight / day.

7. The method according to any one of claims 1-6, wherein the neem extract enriched in azadirachtin A does not comprise neem oil.

8. The method of any one of claims 1-7, wherein a pest control agent is provided to the pests in the fish feed in an amount sufficient to modify the behavior of the pests.

9. The method of any one of claims 1-8, wherein modulating the behavior of the pest comprises one or more of a change in feeding habits, a change in feeding patterns, a change in appetite, a change in movement patterns, or a change in mating patterns compared to pests found on control animals not fed a pest control agent.

10. The method of claim 9, wherein the change in eating pattern comprises a decrease in eating pattern.

11. The method of claim 9, wherein the change in appetite comprises a decrease in appetite.

12. The method of claim 9, wherein the change in movement pattern comprises a reduction in movement.

13. The method of claim 9, wherein the alteration in mating pattern comprises a reduction in mating pattern.

14. The method of claim 13, wherein the alteration in mating patterns comprises a reduction in the number of live pests present on the fish or in the fish's habitat as compared to pests found on control fish not fed the pest control agent.

15. The method of any one of claims 1-14, wherein the pest control composition is applied to one or more fish during an infection or infestation period for a period of 1 to 20 days.

16. A method for reducing, preventing or controlling a non-copepod parasite infection or infestation in a fish population, the method include: Providing a fish feed comprising a neem extract enriched with azadirachtin A, wherein the neem extract comprises 15 wt % to 33 wt % of azadirachtin A; administering the fish feed comprising the neem extract enriched in azadirachtin A to one or more fish; and wherein the fish feed provides a concentration of 0.01 mg to 5.0 mg azadirachtin A / kg body weight / day to the one or more fish.

17. The method of claim 16, wherein the non-copepod parasite infection or infestation is caused by one or more parasitic organisms, the one or more parasitic organisms comprising Giardia ovale, Centrocera, Cryptocaryon irritans, Scima spp., Trichodina spp., Myxosporea salmoninae, Ichthyophthirius multifida, Kudaoa serrata, Cerebral iodine bubble worm, Neoparamoebae, Saprolegnia parasiticus, Monogenea, Trichodina spp., or Arugam ceylon leech.

18. The method of claim 16, wherein the fish feed is administered to farmed fish for at least 11 days.

19. The method of claim 16, wherein the fish feed is administered to farmed fish for at least 14 days.

20. The method of claim 16, wherein the concentration of azadirachtin A comprises a concentration in an amount effective to increase the efficacy of azadirachtin A against non-copepod parasite infection or infestation compared to fish fed a diet lacking the azadirachtin A-enriched neem extract.

21. The method of claim 16, wherein the neem extract enriched in azadirachtin A is administered to the fish at a concentration of 1.5 mg to 2.5 mg azadirachtin A / kg body weight / day.

22. The method of claim 16, wherein the neem extract enriched in azadirachtin A is administered to the fish at a concentration of 2.6 mg to 5.0 mg azadirachtin A / kg body weight / day.

23. The method of claim 16, wherein the fish feed further comprises one or more components comprising an antibacterial, antifungal, antiviral, antiparasitic, or antiprotozoal agent.

24. The method of claim 16, wherein the fish feed is administered to fish species belonging to one or more families including: Cyprinidae, Cichlidae, Panfish, Sciaenidae, Serranidae, Carangidae, Sparidae, Perchidae, Lycoperchidae, Mugilidae, Cypriniformes, Percidae, Channaidae, Tilapia, and Salmonidae.

25. The method of claim 16, wherein the pest control agent is configured to produce an inhibitory effect comprising one or more of an antiparasitic effect, an antibacterial effect, an antiviral effect, an antifungal effect, or an antiprotozoal effect.

26. The method of claim 16, wherein the neem extract enriched in azadirachtin A does not comprise neem oil.

27. The method of claim 16, wherein the pest control agent is provided to the pest in the fish feed in an amount sufficient to modify the behavior of the pest.

28. The method of claim 16, wherein modulating the behavior of the pest comprises one or more of: a change in feeding habits, a change in feeding patterns, a change in appetite, a change in movement patterns, or a change in mating patterns as compared to pests found on control animals not fed a pest control agent.

29. The method of claim 28, wherein the change in eating pattern comprises a decrease in eating pattern.

30. The method of claim 28, wherein the change in appetite comprises a decrease in appetite.

31. The method of claim 28, wherein the change in movement pattern comprises a reduction in movement.

32. The method of claim 28, wherein the alteration in mating pattern comprises a reduction in mating pattern.

33. The method of claim 32, wherein the alteration in mating patterns comprises a reduction in the number of live pests present on the fish or in the fish's habitat as compared to pests found on control fish not fed the pest control agent.

34. The method according to claim 16, wherein the neem extract enriched in azadirachtin A is obtained by a process comprising the steps of: Neem seeds are available; crushing the neem seeds; Extract azadirachtin from the crushed seeds using water; adding a second extraction solution, the second extraction solution comprising: a non-aqueous solvent that is immiscible with water and has a higher solubility for azadirachtin than water; or a surfactant having a turbidity temperature between 20°C and 80°C; and Concentrated azadirachtin is recovered from the second extraction solution.

35. A method for reducing, preventing or controlling a non-copepod parasite infection or infestation in fish, said method include: Provided is a pest control agent composition comprising a pest control agent, wherein the pest control agent comprises a neem extract rich in azadirachtin A; as well as applying the pest control composition to one or more fish during an infection or infestation period for a period of 1 to 20 days; The concentration of azadirachtin A applied to the fish via the pest control agent composition is about 0.01 mg to 5 mg azadirachtin A / kg body weight / day.

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