COMPOSITIONS AND METHODS FOR CONTROLLING INSECT

PROLIFERATION

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S.

Provisional Application No. 62/029,863, filed on July 28, 2014, which is hereby incorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under ROl DK084186 awarded by the National Institute of Health (NIH). The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Many strategies exist for blocking insect reproduction. However, there is a pressing need to develop an approach to selectively control insect populations without damage to the environment.

SUMMARY OF THE INVENTION

The invention provides a method of preventing, reducing, or inhibiting insect reproduction comprising administering to the insect a composition comprising an agent that inhibits spermatogenesis (e.g., late stage spermatogenesis) in the insect, thereby preventing, reducing, or inhibiting reproduction in the insect. Preferably, the agent that inhibits spermatogenesis in the insect comprises an agent that inhibits expression or activity of iodotyrosine deodinase (IYD) in the insect (i.e., in the male insect). For example, the agent inhibits expression or activity of IYD in a testis of the insect.

The methods described herein reduce a population size of the insect, i.e., the methods described herein reduce/control insect proliferation. For example, the methods described herein reduce an insect population, i.e., the methods reduce/decrease the number of insects in a defined environment by at least 1%, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.

The methods described herein also reduce fecundity and/or fertility of the insect For example, the methods described herein reduce/decrease fecundity and/or fertility of the insect by at least 1%, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or by 100%.

In some cases, the agent for inhibiting expression or activity of IYD comprises an antibody or fragment thereof, a binding protein, a polypeptide, or any combination thereof.

In other cases, the agent for inhibiting expression or activity of IYD comprises a small molecule. A small molecule is a compound that is less than 2000 Daltons in mass. The molecular mass of the small molecule is preferably less than 1000 Daltons, more preferably less than 600 Daltons, e.g., the compound is less than 500 Daltons, less than 400 Daltons, less than 300 Daltons, less than 200 Daltons, or less than 100 Daltons.

Small molecules are organic or inorganic. Exemplary organic small molecules include, but are not limited to, aliphatic hydrocarbons, alcohols, aldehydes, ketones, organic acids, esters, mono- and disaccharides, aromatic hydrocarbons, amino acids, and lipids.

Exemplary inorganic small molecules comprise trace minerals, ions, free radicals, and metabolites. Alternatively, small molecules can be synthetically engineered to consist of a fragment, or small portion, or a longer amino acid chain to fill a binding pocket of an enzyme. Typically small molecules are less than one kilodalton.

Suitable small molecule inhibitors include 4-OH-2',3,4',5,6'-PCB; 4-OH-2',3,4',6'- TCB; 4-OH-2,2',5,5'-TCB; 4,4'-diOH-3,3',5,5'-TCB; 3-OH-2,2',5,5'-TCB; 4'-OH-BDE-17; 4-OH-BDE-42; 4'-OH-BDE-49; 4-OH-BDE90; 2-OH-BDE-15; 2'-OH-BDE-28;

Bromoxynil; Oxyclozanide; Tribromsalan; Closantel; Triclosan; Bithionol; Nitroxynil;

Benzbromarone Rose Bengal; Erythrosine B; Phloxine B; D,L-3-(2-pyridon-5-yl)alanine; and D,L-3-(N-methyl-2-pyridon-5-yl)alanine; D,L-3-(N-ethyl-2-pyridon-5-yl)alanine; and D,L-3- (N-isopropyl-2-pyridon-5-yl)alanine. In a preferred aspect, the small molecule inhibitor is L- 3-(N-methyl-2-pyridon-5-yl)alanine.

In other aspects, the agent comprises a nucleic acid molecule. For example, the nucleic acid molecule comprises double stranded ribonucleic acid (dsRNA), small hairpin RNA or short hairpin RNA (shRNA), or antisense RNA, or any portion thereof.

A variety of administration routes are available. For example, the agent is administered topically, orally, via inhalation, or via injection.

The composition is in the form of a gas, a liquid, a solid, or a semi-solid. In a preferred aspect, the composition comprises sugar water. In another example, the agent is administered as a gaseous or liquid spray. In some cases, the agent is soluble in water.

Preferably, the agent is nontoxic.

The effective amount of the agent is from 0.001 mg/kg to 250 mg/kg body weight, e.g., 0.001 mg/kg, 0.05 mg/kg 0.01 mg/kg, 0.05mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 225 mg/kg, or 250 mg/kg body weight.

In some cases, the agent is administered at least once per day, at least once per week, or at least once per month. The agent is administered for a duration of one day, one week, one month, two months, three months, six months, 9 months, or one year. For example, the compositions described herein are placed in an insect "trap" as food for ingestion by the insects. The "traps" are replenished with fresh composition as needed, e.g., at least once per day, at least once per week, at least once per month, or at least once every three moths.

The compositions and methods described herein are useful in inhibiting reproduction in any insect. For example, suitable insects include cockroaches (e.g., brown-banded cockroaches, German cockroaches, American cockroaches, Oriental cockroaches), beetles (woodworms, death watch beetles, fur beetles, varied carpet beetles, spider beetles, mealworm beetles, centipedes, red flower beetle, pine beetle), earwigs, flies (e.g., bottle flies, blue bottle flies, green bottle flies, house flies, fruit flies, horse flies, black flies, deer flies, house flies, teste flies), cicadas, hoppers, ants (e.g., Argentine ants, carpenter ants, fire ants, odorous house ants, pavement ants, pharaoh ants, thief ants, a leafcutter ant, a mountain fire ant, a Jerdon't jumping ant), aphids, bed bugs, bees (e.g., a common eastern bumble bee, a bumble bee, a honeybee, an alfalfa leafcutter bee), wasps, mantids, earthworms, terrestrial crabs, firebrats, fleas, snails, slugs, crickets (e.g., house crickets), hornets, locusts, lice, moths (e.g., almond moths, Indianmeal moths, cloths moths, gypsy moths (common clothes moths), brown house moths), red spiders, silverfish, woolice, termites (e.g., dampwood termites, subterranean termites), ticks, wasps, mosquitos, gnats, millipedes, stink buds, weevils, mites, silk worm, daphnia, fleas, Assassin bugs (kissing bugs), eye gnats, or rat fleas and woolly bears (e.g., hairy caterpillar).

The compositions and methods are also useful in inhibiting reproduction in the following insects: Culex quinquefasciatus (Southern house mosquito), Drosophila willistoni (Fruitfly), Drosophila mojavensis (Fruitfly), Drosophila pseudoobscura (Fruitfly),

Drosophila grimshawi (Fruitfly), Drosophila virilis (Fruitfly), Drosophila ananassae (Fruitfly), Ceratitis capitata (Mediterranean fruitfly), Drosophila sechellia (Fruitfly), Drosophila persimilis (Fruitfly), Drosophila simulans (Fruitfly), Drosophila inelanogasler (Fruitfly), Anopheles darling (Mosquito), Anopheles gambiae (Mosquito), Musca domestica (Housfly), Drosophila erecta (Fruitfly), Drosophila yakuba (Fruitfly), Aedes aegypti (Mosquito, yellow fever), Bombyx mori (Silk worm), Tribolium castaneum (red flour beetle), Nasonia vilripennis (wasp), Apis mellifera (honeybee), Apis dorsata (honeybee), Megachile rotundata (alfalfa leafcutter bee), Dendroctonus ponderosae (mountain pine beetle), Bombus impatiens (common eastern bumble bee), Bombus terrestris (bumble bee), Acromynnex echinatior (leafcutter ants), Solenopsis invicta (fireant), Apis florea (honeybee),

Harpegnathos saltator (Jerdon's jumping ant), Cerapachys biroi (ant), Daphnia pulex (daphnia), Camponotus floridanus (ant), and Pediculus hunianus corporis (lice).

In some cases, the methods described herein are useful for inhibiting the reproduction of any invertabrate, such as a crab, a lobster, a snail, a clam, an octopus, a starfish, asea-urch or a worm.

In one aspect, the compositions are administered within a device, e.g., a "trap," that attracts an insect to the device in order to administer the compositions described herein to inhibit spermatogenesis within the insect

Definitions

By "agent" is meant any small molecule compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.

"Controlling pests" as used in the present invention means killing pests, or preventing pests from developing, or from growing or preventing pests from infecting or infesting. Controlling pests as used herein also encompasses controlling insect progeny (development of eggs). Controlling pests as used herein also encompasses inhibiting viability, growth, development, or reproduction of the insect, or decreasing pathogenicity or infectivity of the insect

"Detect" refers to identifying the presence, absence, or amount of the nucleic acid (e.g., RNA) to be detected. By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via, for example, spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels may include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

By "effective amount" is meant the amount of an agent required to inhibit reproduction in an insect.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

"Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

By "inhibitory nucleic acid" is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Chitosan compositions are useful for the delivery of

polynucleotides, such as inhibitory nucleic acid molecules. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.

By "isolated polynucleotide" is meant a nucleic acid (e.g., RNA, DNA, cDNA, etc.) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By "Kd" is meant dissociation constant, a parameter of a drug that characterizes the specificity of interaction between a drug and its target

By "k _cat " is meant the turnover number for an enzyme; the number of substrate molecules each enzyme site converts to product per unit time.

By "Km" is meant Michaelis-Menten constant, which represents the substrate concentration at which an enzymatic reaction rate is half of Vmax.

By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

By "modulate" is meant alter (increase or decrease). Such alterations are detected by standard art known methods such as those described herein.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent.

As used herein, the terms "prevent," "preventing," and "prevention," refer to reducing or inhibiting reproduction in an insect. "Primer set" means a set of oligonucleotides that may be used, for example, for PCR. A primer set would consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, "nested sub-ranges" that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

By "reduces" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or

100%.

By "reference" is meant a standard or control condition.

A "reference sequence" is a defined sequence used as a basis for sequence comparison or a gene expression comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 40 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 or about 500 nucleotides or any integer thereabout or there between.

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison. Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFTT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;

aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.

In some cases, by "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

Alternatively, by "subject" is meant an insect

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The transitional term "comprising," which is synonymous with "including,"

"containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase

"consisting of excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel

characteristic(s)" of the claimed invention.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic showing the reductive deiodination of mono- and

diiodotyrosine catalyzed by IYD (iodotyrosine deiodinase) and its bound cofactor, flavin mononucleotide (FMN and in its reduced form, FMNH ₂ ).

Figure 2A and Figure 2B are schematics showing the co-crystal structure of mouse IYD-MIT (monoiodotyrosine). Figure 2A is a molecular model of the α ₂ dimer of mmlYD- ΜΓΓ (mouse iodotyrosine deiodinase). The monomers are depicted in green and purple cartoon representation. The FMN (Flavin mononucleotide) and MIT molecules are shown in stick representation and colored according to atom. Figure 2B is a molecular model of the α ₂ dimer of mmlYD-MIT showing the residues involved in substrate recognition, Y157, E153 and K178. T235 provides an important hydrogen bond to the N5 of the FMN.

Figure 3 is a multiple sequence alignment showing that IYD homologs are present in diverse organisms. Determinants of IYD are outlined in red, residues interacting with FMN are marked with (+), residues interacting with substrate are marked with (*), residues marked in red are fully conserved, lid-forming sequences are boxed, and secondary structural elements derive from the structure of mmlYD-MIT. Numbering of the amino acids for each protein is indicated on the left and right of the alignment. Secondary structure elements are derived from the crystal structure of mmlYD bound to DIT (diiodotyrosine). Figure 4 is a diagram depicting the phylogenic analysis of catalytic domains. All proteins containing the signature residues (E1S3, Y1S7 and K178) promote deiodination of DIT. The closest structural analog (Blub) lacks these residues, and does not support deiodination of DIT (diiodotyrosine). The branch lengths are proportional to the number of amino acid substitutions per aligned residue.

Figure 5 is a graph showing the expression of IYD mRNA in Drosophilia. Tissue specific mRNA expression profiling for Drosophila melanogaster (fly) indicated very high levels if IYD (gene CG6279 in flybase) in the adult male testes. Adult Drosophila are shown in red bars, and larva Drosophila are shown in blue bars.

Figure 6A and Figure 6B is a diagram and a bar graph, respectively, showing the RNAi knockdown of IYD in Drosophila testis. Figure 6A is a diagram showing the promoter dependent knockdown of IYD mRNA. Figure 6B is a bar graph showing the fertility and fecundity of eya-GAL4, UAS-dmlYD-RNAi and eya-CAL1/UAS-dmYD-RNAi.

Figure 7A, Figure 7B, and Figure 7C is a diagram, a bar graph, and a

photomicrograph, respectively, showing that late stage development of Drosophila is sensitive to IYD. Figure 7A is a diagram showing the stages of spermatogenesis in

Drosophila. Figure 7B is bar graph showing the abnormalities detected in the testis after RNAi degradation of IYD mRNA. Figure 7C is an image showing the phase contrast of drosophila testes. Suppression of dmIYD disrupts late stage spermatogenesis.

Figure 8 is a bar graph showing the male germline cell specific expression profile of CG6279. Germline and somatic stem cells were located in niche, GB in gonioblast, S4 through S16 were spermatogonia (pre-differentiation), EC 16 onwards were spermatocytes post-differentiation.

Figures 9A, Figure 9B, Figure 9C, and Figure 9D are photomicrographs showing testes of flies treated with digoxigenin-labeled riboprobes. Figure 9A is an image showing CG6279 isoform A specific antisense riboproboe. Figure 9B is an image showing CG6279 isoform A+B specific antisense riboprobe. Figure 9C is an image showing the control where isoform A specific sense riboprobes were used. Figure 9D is an image showing the control where isoform A + B specific sense riboprobes were used. (*) indicates early stages of spermatogenesis. (**) indicates later stages of spermatogenesis with strong staining for hybridization of riboprobes with CG6279 mRNA.

Figure 10 is a diagram showing the HPLC method schematic (left to right) for separation, detection and quantification of tyrosine produced from ΜΓΓ. All gradients were linear over the indicated time. A drop in the B mobile phase from 60% to30% over 2 mins after the analytical time prevented impurities from affecting the internal standard peak by delaying their elution. The column was washed with 95% B and equilibrated with A prior to next injection of analyte.

DETAILED DESCRIPTION OF THE INVENTION

As described in detail below, described herein are compositions and methods that are useful for inhibiting spermatogenesis in insects, e.g., drosophila, by decreasing the expression or activivty of iodotyrosine deiodinase (IYD). A putative IYD from Drosophila (dmIYD) was expressed and confirmed to catalyze deiodination. Its k _cat /K _m value was similar to those of all other homologues tested. Expression of dmIYD was primarily limited to the testis and its suppression by RNAi significantly disrupted spermatogenesis, particularly within the late stages of sperm development. The enzyme was critical for spermatogenesis in insects, but not mammals. Fertility and fecundity were greatly reduced when expression of this enzyme was suppressed, and hence, proliferation of insects is selectively reduced by inhibiting this enzyme. The molecular basis was consistent with iodotyrosine acting as a cellular signal and representing a progenitor of thyroxine. Prior to the invention described herein, a biological role had not been previously identified for this enzyme.

Iodotyrosine (IYD) deiodinase

IYD has been traditionally associated with iodide conservation by generating iodide (I-) from mono- and diiodotyrosine. IYD facilitates iodide salvage in thyroid tissue in vertebrates by catalyzing deiodination of mono- (I-Tyr) and diiodotyrosine (DIT, I ₂ -Tyr), that are formed as halogenated byproducts of thyroid hormone biosynthesis (often in surprisingly large quantities) (Goswami, A.; Rosenberg, I. N., Endocrinology 1977, 101, 331-341, Gnidehou, S.et al., FASEB J. 2004, 18, 1574-1576 and Rokita, S. E. et al., Biochimie 2010, 92, 1227-1235). Prior to the invention described herein, IYD had remained poorly characterized for more than 50 years despite its significant role in intrathyroidal iodine metabolism. Patients with deficient IYD suffer from goiter, enlarged thyroid, and other symptoms associated with hypothyroidisim because iodotyrosine cannot be directly reutilized for thyroid hormone synthesis. The dehalogenation reaction catalyzed by IYD is unusual for aerobic organisms since the carbon-iodine bond is broken through a reductive process. More commonly, dehalogenation is accomplished by hydrolytic or oxidative pathways. Many of the bacterial enzymes responsible for dehalogenation have emerged from existing enzyme superfamilies.

IYD has been associated with iodide homeostasis in humans since its mutation, first discovered over SO years ago, was found to limit thyroid function. More recently, sequence homology and structural data identified this enzyme as the only known representative of the nitro-FMN reductase structural superfamily in humans. Members of this superfamily primarily derive from bacteria and their activities range from generating and chaperoning dihydroflavin, FADH ₂ (Flavin Reductase Protein, FRP), to reducing nitroaromatic compounds (NOX) and degrading flavin for vitamin B ₁₂ biosynthesis (BluB). All IYDs are indicated by four key residues, a threonine (Thr) for hydrogen bonding to the NS position of FMN, a glutamic acid (Glu), tyrosine (Tyr) and lysine (Lys) for coordinating to the substrate iodotyrosine.

Functional IYDs have been confirmed in Chordata, Arthropoda, Cnidaria, Eubacteria and Archaeabacteria. Chordata require the tetraiodinated hormone, thyroxine, and rely on IYD to salvage iodide from mono- and diiodotyrosine formed as byproducts of its biosynthesis. Thyroxine is not produced by invertebrates and thus IYD is unlikely to provide iodide for its synthesis. Bacteria may use IYD in a catabolic role since this enzyme is capable of dehalogenating chloro, bromo- and iodotyrosines. As described herein, the function of IYD in Arthropoda and Cnidaria is examined in the model Arthropoda,

Drosophila melanogaster.

Drosophila IYD

As described in detail below, this Drosophila gene acts as an iodotyrosine deoidinase and is critical for sperm formation in insect testes. Prior to the invention described herein, no evidence in the literature had made a connection between the catalytic activity of this enzyme and spermatogenesis. As described herein, the deiodinase gene was suppressed, and a loss in Drosophila fertility was observed. This organism is a well-studied model for insects.

Two isoforms have been reported for Drosophila IYD (dmIYD) in the NCBI databases. These two are related by alternative splicing of the CG6279 gene. Isoform B resembles IYD orthologs from other organisms and includes a membrane domain, an intermediate domain and a catalytic domain (Phatarphekar, A. et al, Mol. Biosys. 2014, 10, 86-92 and Friedman, J. E. et al. J. Biol. Chem. 2006, 281, 2812-2819). Isoform A is unique to the Drosophila genus by the added presence of a large N-terminal domain (470 amino acids) that does not coincide with any known protein domains listed in the conserved domain database (CDD).

The amino acid sequence of Drosophila melanogaster IYD (A Isoform) is shown below:

herein by reference.

The nucleic acid sequence of Drosophila melanogaster IYD (A Isoform) is shown below:

herein by reference.

The amino acid sequence of Drosophila melanogaster IYD (B Isoform) is shown below:

1 mdvdelisss kllkhwpslf itlaliwivk rlffkgnrvl ktynldegye eevehf adlg

61 delgpaledk phvpfvpgqn lnpngakrly elmrgrrsir sfnshpkpdl sviedciraa 121 gtapsgahte pwtycwqep elkrsireiv eqeelvnysq rmhpqwvtdl rplqtnhvke 181 ylteapylil ifkqtyglse ngkrmrrhyy neistsiaag illcalqaag laslvttpln 241 cgpalrnllg rpvnekllil lpvgypkdgc tvpdlarknl snimvtf

(SEQ ID NO: 3) GenBank Accession No: NP_001163414 (Version 1), incorporated herein by reference.

The nucleic acid sequence of Drosophila melanogaster IYD (B Isoform) is shown below:

(SEQ ID NO: 4) GenBank Accession No: NM_001169943 (Version 1), incorporated herein by reference.

Human IYD

Iodotyrosine deiodinase (IYD) is a membrane bound enzyme in humans that is primarily expressed in thyroid tissue to recover iodide from mono- and diiodotyrosine (MIT, DIT) generated during thyroxine biosynthesis. This enzyme is highly unusual for its (i) ability to promote reductive dehalogenation and (ii) reliance on flavin to catalyze this process (Figure 1). In vivo, NADPH appears to act as the electron donor for this reaction but IYD is not directly reduced by NADPH. In vitro, dithionite serves as an alternative electron donor. IYD also effects debromination and dechlorination of the corresponding halotyrosines. A review on the enzyme iodotyrosine deiodinase focusing on its role in mammals is found in Rokita, S.E. et al., Biochimie 2010, 92, 1227-1235, incorporated herein by reference. A review on the rare occurrence of reductive dehalogenases in aerobic organisms is found in Rokita, S.E., Handbook in Flavoproteins, vol. 1 DeGruyter Berlin, 2013. P. 337-350, incorporated herein by reference.

The amino acid sequence of Human IYD is shown below:

reference.

The nucleic acid sequence of Human IYD is shown below:

(SEQ ID NO: 6) GenBank Accession No: NM_203395 (Version 2), incorporated herein by reference.

The amino acid sequence of mouse (mus musculus) IYD is shown below:

reference.

The nucleic acid sequence of mouse (mus musculus) IYD is shown below:

reference.

The amino acid sequence of porcine (Sus scrofa) IYD is shown below:

The nucleic acid sequence of porcine (Sus scrofa) IYD is shown below:

reference.

The amino acid sequence of Honeybee IYD is shown below:

herein by reference.

herein by reference.

The amino acid sequence of Mosquito (Anopheles darling) IYD is shown below:

herein by reference.

incorporated herein by reference.

The amino acid sequence of Silk Worm (Bombyx mori) IYD is shown below:

herein by reference.

The nucleic acid sequence of Silk Worm (Bombyx mori) IYD is shown below:

incorporated herein by reference.

The amino acid sequence of a wasp (Nasonia vitripennis) IYD is shown below:

301 vpglkrkdld evlvefe

(SEQ ID NO: 17) GenBank Accession No: XP .008213524 (Version 1), incorporated herein by reference.

The nucleic acid sequence of a wasp (Nasonia vitripennis) IYD is shown below:

incorporated herein by reference.

Another exemplary amino acid sequence of mosqiuto {Anopheles gambie) IYD is shown below:

herein by reference.

Another exemplary nucleic acid sequence of mosquito {Anopheles gambie) IYD is shown below:

(SEQ ID NO: 20) GenBank Accession No: XM_315442( Version 4), incorporated herein by reference.

The amino acid sequence of a house fly (Musca domestica) IYD is shown below:

herein by reference.

The nucleic acid sequence of a house fly {Musca domestica) IYD is shown below:

herein by reference.

IYD Small molecule inhibitors

Several small molecule inhibitors have shown IYD-inhibitory activity. As such, these small molecules are useful as inhibitors of various IYDs. In humans, these inhibitors can disrupt thyroid hormone homeostasis by blocking iodide recycling through inhibition of IYD activity in the thyroid. Various classes of phenolic compounds comprise: PCBs (polychlorinated biphenyls), PBDEs (polybrominated diphenyl ethers), agrochemicals, antiparasitics, pharmaceuticals and food colorants. PCB examples include:

PBDE examples include:

Agrochemical examples include:

Antiparasitic examples include:

Food colorant examples include:

Competitive IYD small molecules

The pyridonyl amino acids (Table 6) were all observed to be reversible and competitive inhibitors of diiodotyrosine turnover under standard assay conditions ( unishima, M., et al, J. Am. Chem. Soc. 1999, 121, 4722-4723, incorporated herein by reference).

Table 6: Kinetic and binding constants for iodotyrosine deiodinase.

Spermatogenesis

The method described herein comprise administering to the insect a composition comprising an agent that inhibits spermatogenesis (e.g., late stage spermatogenesis) in the insect, thereby preventing, reducing, or inhibiting reproduction in the insect

Spermatogenesis is the process in which spermatozoa are produced from male primordial germ cells by way of mitosis and meiosis. The initial cells in this pathway are called spermatogonia, which yield primary spermatocytes by mitosis. The primary spermatocyte divides meiotically, Meiosis I, into two secondary spermatocytes; each secondary spermatocyte divides into two spermatids by Meiosis II. These develop into mature spermatozoa, also known as sperm cells. Thus, the primary spermatocyte gives rise to two cells, the secondary spermatocytes, and the two secondary spermatocytes by their subdivision produce four spermatozoa.

Spermatogenesis begins immediately after meiosis, when spermatids begin to undergo the morphological changes required for sperm development. Elongation of the spermatids occurs within a syncytial cyst that becomes polarized such that all the nuclei localize to one end and the growing ends of the sperm tails are found at the other end. Spermatid elongation is accompanied by dramatic changes in nuclear shape. Following meiosis, round spermatids (alternatively, onion stage spermatids), contain spherical nuclei that are approximately 5 μm in diameter. During sperm development, the nuclei become thinner and the chromatin condenses. Following elongation and nuclear shaping, the mature sperm are invested with their own membranes in a process called individualization. Individualization requires formation of an individualization complex (IC), composed of actin cones that form around the nuclei in a mature spermatid cyst. Following individualization, each group of mature sperm becomes coiled in a process that brings the entire length of the nearly 2mm long sperm bundle into the base of the testis. After coiling, the sperm are released into the testis lumen and are transferred to the seminal vesicle, where they are stored until needed for fertilization. Common insects

Insects are a class of invertebrates within the arthropod phylum that have an achitinous exoskeleton, a three-part, body (head, thorax and abdomen), three pairs of jointed legs, compound eyes and one pair of antennae. They are among the most diverse groups of animals on the planet, including more than a million described species and representing more than half of all known living organisms. The exact number of species is estimated between six and ten million, and potentially represents over 90% of the differing animal life forms on E arth. Insects may be found in nearly all environments, although only a small number of species reside in the oceans, a habitat dominated by another arthropod group, crustaceans.

An arthropod is an invertebrate animal having an exoskeleton (external skeleton), a segmented body, and jointed appendages. Arthropods form the phylum Arthropoda, and include the insects, arachnids, myriapods, and crustaceans. Arthropods are characterized by their jointed limbs and cuticle made of chitin, often mineralised with calcium carbonate. The arthropod body consists of segments, each with a pair of appendages. The rigid cuticle inhibits growth, so arthropods replace it periodically by moulting. Their versatility has enabled them to become the most species-rich members of all ecological guilds in most environments. They have over a million described species, making up more than 80% of all described living animal species, some of which, unlike most animals, are very successful in dry environments. Like their exteriors, the internal organs of arthropods are generally built of repeated segments. Their nervous system is "ladder-like", with paired ventral nerve cords running through all segments and forming paired ganglia in each segment Their heads are formed by fusion of varying numbers of segments, and their brains are formed by fusion of the ganglia of these segments and encircle the esophagus. The respiratory and excretory systems of arthropods vary, depending as much on their environment as on the subphylum to which they belong.

Extrapolation of the data herein indicates that inhibiting I YD enzyme in insects (e.g., mosquitos), may provide a strategy for population control. The compositions and methods described herein are useful for inhibiting spermatogenesis in any insect, e.g., cockroaches (e.g., brown-banded cockroaches, German cockroaches, American cockroaches, Oriental cockroaches), beetles (woodworms, death watch beetles, fur beetles, varied carpet beetles, spider beetles, mealworm beetles, centipedes, red flower beetle, pine beetle), earwigs, flies (e.g., bottle flies, blue bottle flies, green bottle flies, house flies, fruit flies, horse flies, black flies, deer flies, house flies, teste flies), cicadas, hoppers, ants (e.g., Argentine ants, carpenter ants, fire ants, odorous house ants, pavement ants, pharaoh ants, thief ants, a leafcutter ant, a mountain a fireant, a Jerdon't jumping ant), aphids, bed bugs, bees (e.g., a common eastern bumble bee, a bumble bee, a honeybee, an alfalfa leafcutter bee), wasps, mantids, earthworms, terrestrial crabs, firebrats, fleas, snails, slugs, crickets (e.g., house crickets), hornets, locusts, lice, moths (e.g., almond moths, Indianmeal moths, cloths moths, gypsy moths (common clothes moths), brown house moths), red spiders, silverfish, woolice, termites (e.g., dampwood termites, subterranean termites), ticks, wasps, mosquitos, gnats, millipedes, stink buds, weevils, mites, silk worm, daphnia, fleas, Assassin bugs (kissing bugs), eye gnats, or rat fleas and woolly bears (e.g., hairy caterpillar).

The compositions and methods are also useful in inhibiting reproduction in the following insects: Culex quinquefasciatus (Southern house mosquito), Drosophila willistoni( Fruitfly), Drosophila mojavensis (Fruitfly), Drosophila pseudoobscura (Fruitfly),

Drosophila grimsfiawi (Fruitfly), Drosophila virilis (Fruitfly), Drosophila anariassae (Fruitfly), Ceratitis capitata (Mediterranean fruitfly), Drosophila sechellia (Fruitfly), Drosophila persimilis (Fruitfly), Drosophila simulans (Fruitfly), Drosophila tnelanogaster (Fruitfly), Anopheles darling (Mosquito), Anopheles gambiae (Mosquito), Musca domestica (Housfly), Drosophila erecta (Fruitfly), Drosophila yakuba (Fruitfly), Aedes aegypti (Mosquito, yellow fever), Bombyx mori (Silk worm), Tribolium castaneum (red flour beetle), Nasonia vitripennis (wasp), Apis mellifera (honeybee), Apis dorsata (honeybee), Megachile rotundata (alfalfa leafcutter bee), Dendroctonus ponderosae (mountain pine beetle), Bombus impatiens (common eastern bumble bee), Bombus terrestris (bumble bee), Acromyrmex echinalior (leafcutter ants), Solenopsis invicla (fireant), Apisflorea (honeybee),

Harpegnathos saltator (Jerdon 's jumping ant), Cerapachys biroi (ant), Daphnia pulex (daphnia), Camponotus floridanus (ant), and Pediculus humanus corporis (lice).

Blocking insect reproduction

The methods described herein offer a unique and selective approach for controlling insect population without damage to the environment Inhibiting this enzyme in insects, (e.g., mosquitos) provides a new strategy for insect population control, i.e., reducing the number of insects.

Also provided is the identification of chemical, e.g., small molecule, inhibitors of IYD. The methods described herein involve selective application of an agent within the environment to selectively target insects (e.g., mosquitos) for controlling insect population, i.e., reducing the number of insects. Alternatively, the agent may be selectively placed such that the insect of interest is the only organism susceptible to population control.

One means of population control in insects is an insect growth regulator (IGR), which is a chemical substance that inhibits the life cycle of an insect, and is commonly used to control populations of harmful pests. IGRs prevent an insect from reaching maturity by interfering with its molting process (for example, azadirachtin, hydroprene, methoprene, pyriproxyfen and tritlumuron). This results in immature insects that cannot reproduce. Hormonal IGRs mimic or inhibit one of the two major hormones involved in insect molting. Chitin synthesis inhibitors prevent the formation of chitin, a carbohydrate used to form the insect's exoskeleton. As described herein, in some cases, the compositions of the invention are administered in conjunction with IGRs.

Additionally, insecticides are substances that are used to kill insects, and are used in agriculture, medicine, industry, and by consumers. Two major groups include systemic and contact insecticides. Systemic insecticides become incorporated and distributed systemically throughout the whole plant. When insects feed on the plant, they ingest the insecticide. Systemic insecticides have activity pertaining to their residue which is called "residual activity" or long-term activity. Contact insecticides are toxic to insects upon direct contact. These can be inorganic insecticides, which are metals and

include arsenates, copper and fluorine compounds, which are less commonly used, and the commonly used sulfur. Contact insecticides can be organic insecticides, i.e. organic chemical compounds, synthetically produced, and comprising the largest numbers of pesticides used today, or, they can be natural compounds like pyrethrum, neem oil etc. Contact insecticides usually have no residual activity. Some insecticides kill or harm other organisms in addition to those they are intended to kill. For example, birds may be poisoned when they eat food that was recently sprayed with insecticides or when they mistake an insecticide granule on the ground for food and eat it.

Dosages and frequency of administration of pest-controlling agent

The effective amount of the agent is from 0.001 mg/kg to 250 mg/kg body weight, e.g., 0.001 mg/kg, 0.005 mg/kg, 0.01 mg/kg, 0.05 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 225 mg/kg, or 250 mg/kg body weight. The agent is more effective with some insects than others. Amounts of some of the agents may be as low as 2.5 milligrams per kilogram of body weight, and possibly less. Even smaller amounts, for instance as little as 0.05 milligrams per kilogram of body weight are effective when the dosage is continued from day to day as may be desirable in some circumstances.

One treatment (dosage) may also be sufficient to alter such transient insects as mosquitoes and flies. Within a few weeks or months after treatment, it may become apparent that repeated dosages may be required to control insects, e.g., mosquitoes over a long period of time. As such, this type of insect control may be desired in particular circumstances over long periods of time. IYD inhibitors are not. contact toxins, i.e., adult insects exposed to IYD inhibitors will likely not die; however, their reproduction will be blocked. Male mosquitos live ~ 5 days and females can live for weeks, thus, the results of treatment may take days to weeks to detect in mosquitos. The effect depends on the lifetime of each target insect.

Insect traps

The In2Care® Mosquito Trap uses a unique mixture of bio-actives that target not only the mosquito and its larvae, but can also have an impact on the Dengue virus inside the mosquito. Trap activity is not limited to the trap itself, but extends to the surrounding area due to its mosquito-driven larvicide spreading features. The trap actives provide an environmentally-friendly alternative to synthetic pesticides and combines active ingredients that individually are used in other mosquito products which are WHO recommended and/or US-EPA approved.

Similar traps are utilized in the compositions and methods described herein. For example, the compositions are administered within a device, e.g., a "trap," that attracts an insect to the device in order to administer the compositions described herein to the insect.

In one case, an egg-laying female is attracted to the trap, where it picks up biological agents within the trap to prevent, inhibit, or reduce insect reproduction in a male insect. In other cases, a male insect is attracted to the trap, where it picks up biological agents within the trap to prevent, inhibit, or reduce insect reproduction. Preferably, the agent inhibits spermatogenesis (e.g., late stage spermatogenesis). For example, the agent that inhibits spermatogenesis in the insect comprises an agent that inhibits expression or activity of IYD in the insect, e.g., the agent inhibits expression or activity of IYD in a testis of the insect

EXAMPLES

Example 1: Protein co-crystal structure of mouse IYD in complex with monoiodotyrosine (IYD-MIT) identified key amino acids

The structure of IYD from mouse and human identified key amino acids that were used to query databases of genomic information to identify the presence of the IYD gene in other organisms (Phatarphekar, A. et al., Mol. Biosys. 2014, 10, 86-92). The co-crystal structure of mouse IYD-MIT showed that E153, Y157 and K178 were key residues for substrate recognition (Figure 2A and Figure 2B). T23S provided an important hydrogen bond to the N5 of FMN (Flavin mononucleotide) (Figure 2B). These residues provided markers to identify IYD homologs through the tree of life. The key interacting residues, E153, Y157 and K178 were located on one monomer of the dimer, whereas the FMN molecule was positioned between the monomers, and the adenine tail further extended into the adjacent monomer. Hydrophobic interactions between the phenyl of the ΜΓΓ (monoiodotyrosine) and the flavin rings of FMN may contribute to the overall protein-ligand interaction. The structural results indicated that a small set of residues was sufficient to specify IYD activity. Example 2: IYD Homologs are present in diverse organisms

A sequence alignment between several different organisms (human, mouse, zebrafish, lancelet, drosophilia, daphnia, sea anemone, hydra, bacteria, archaea and bacterial BluB) showed the presence of IYD homologs (Figure 3). All vertebrates and invertebrates sequenced contain IYD. Several key residues were conserved among the organisms. For example, a tryptophan residue in the active site lid, which was responsible for substrate binding. Additionally, a glycine residue within the active site lid is also conserved among the organisms. Determinant IYD residues (glutamic acid, tyrosine, and lysine) were conserved among the organisms and are outlined in red in Figure 3.

Example 3: IYD Exhibits deiodinase activity

Selected orthologs were expressed in K coli and were tested for deiodination activity. All of the orthologs displayed similar catalytic efficiency for deiodination of I ₂ -Tyr with k _cat /K _m values within an order of magnitude of each other (Table 1). However, prior to the invention described herein, the function of halogenated tyrosines and the biological role of IYD was unknown in organisms such as invertebrates that are not know to produce TH. See, Phatarphekar, A., et al., Mol. BioSyst. 2014, 10, 86-92, incorporated herein by reference, for a discussion linking iodotyrosine deiodinase genes with deiodinase activity in organisms not known to require iodide or respond to the iodide-containing hormone thyroxine (including the first insect, honey bee).

All the proteins containing the signature residues (E1S3, Y1S7 and K178) promoted deiodination of DIT (diiodotyrosine), with similar K _m , k _cat and k _cat /K _m values) (Figure 4). For example, the k _cat /K _m in humans was 0.40 ± 0.08 μΜ/min, and 0.56 ±0.09 μΜ/min in drosophilia. The closest structural homolog, BluB (vitamin B _]2 biosynthesis), did not exhibit deiodinase activity (i.e., the K _m for diiodotyrosine was > 133 μΜ). BluB, however, does not contain the signature, key residues (E1S3, Y1S7 and K178). Steady-state kinetic

characterization (K _m , k _cat and k _cat /K _m ) of dehalogenation catalyzed by IYD homologs (intermediate and soluble domains) from representative organisms from diverse phyla is shown in Table 1. Kinetic constants were determined by quantification of ¹²⁵ I- released from [ ¹²⁵ I]-I ₂ -Tyr (Goswami, A.; Rosenberg, I. N., Endocrinology 1977, 101, 331-341, Gnidehou, S.et al., FASEB J. 2004, 18, 1574-1576 and Rokita, S. E. et al., Biochimie 2010, 92, 1227- 1235). Reductive deoidination of iodotyrosine was common to a diverse set of organisms.

Table 1: Kinetic characterization of IYD homologs (intermediate and soluble domains)

aSee, Hu, J.; et al., J. Biol. Chem. 2015, 290, 590-600. "See, Thomas, S. R.et al, J. Biol. Chem. 2009, 284, 19659-19667. °See, Phatarphekar, A. et al., Mol Biosys. 2014, 10, 86-92.

IYD orthologs were present in all 13 species of fruit fly including Drosophila melanogaster that have been sequenced to date. Orthologs of IYD were also found in most all genomes of other insects including the disease carrying vectors such as Anopheles gambiae (malaria) and Aedes aegypti (yellow fever). Using Drosophila as a genetic model, the biological role of IYD in invertebrates was investigated.

Example 4: Tissue distriubtion of IYD in drosophila

The tissue expression of IYD mRNA in Drosophila melanogaster (fly) was disproportinally exressed in the testes as compared to other tissues (e.g., the male accessory gland, the ovaries, the trachea, the heart, and salivary glands) (Figure 5). Tissue specific mRNA expression profiling indicated very high levels of IYD (gene CG6279 in flybase) in adult male testes (Figure 5) (Chintapalli, V. R.; Wang, J.; Nat. Genet. 2007, 39, 715-720). The high IYD expression in the testes was observed in adult drosophilia, as compared to larva which are nearly devoid of IYD. High levels of expression in the testes was surprising, and was further investigated (Figure 8). CG6279 was strongly expressed in testis during later stages of germline development, especially post differentiation (after meiosis).

IYD is a membrane bound enzyme in humans, and is primarily expressed in thyroid tissue. In humans, IYD assists in the recovery of iodide from mono- and diiodotyrosine (MIT, DIT) generated during thyroxine biosynthesis. IYD is crticial for spermatogenesis in insects, but not mammals. Thus, the lack of IYD in mammalian testes makes IYD an attractive target for controlling insect populations without harming mammals, including humans.

Example 5: RNAi knockdown of IYD in Drosophila testis

Selective suppression of a chosen gene in vivo can be performed through applications of RNAi. Application of RNAi in Drosophila provided the first confirmation that IYD was directly involved in spermatogenesis. As described herein, suppression of this gene produced male sterility and developmental anomalies as evident when imaging the testes. Similar effects are expected in other Diptera (e.g., flies, mosquitos, etc.) since all insects sequenced to date contain a gene for IYD. In particular, IYD from Drosophila and Anopheles share 67% identity and 82% similarity.

RNAi-based suppression using double-stranded RNA (dsRNA)

A standard method for inducing RNAi-based suppression in Anopheles is to inject double-stranded RNA (dsRNA) directly into the body cavity. Thus, a series of dsRNAs complementary to the IYD gene are prepared and tested for their ability to suppress IYD in vivo. Not all dsRNA have the same efficiency to suppress the gene and hence multiple dsRNA are tested. If fertility does not diminish with this treatment, the expression of IYD in testes is measured with qPCR to confirm its presence. Additionally, direct injection may not necessarily suppress genes in the testes.

If necessary, dsRNA is also injected along with a fluorescent marker gene into embryos to generate transgenic species with the innate ability to suppress IYD expression. As described herein, a similar strategy was used in Drosophila and placed RNAi suppression of IYD under control of a series of promoters induced at different stages of spermatogenesis. Loss of IYD activity is observed in vivo as a loss of fertility and fecundity. Again, qPCR is used to correlate gene expression with the observed phenotype. Promoter dependent knockdown of IYD

Expression of IYD mRNA was decreased using promotor dependent knockdown (Figure 6A, Figure 6B, and Figure 6C). Promoters used for IYD knockdown are shown in Table 2.

Table 2: Promoters used for IYD knockdown

Fertility,

Promoter Cell Type Expression Fecundity

Stage affected?

nos germ early no bam germ iate no c587 somatic early no

tj somatic early no eya somatic iate yes

As described herein, Drosophila IYD plays a key role in spermatogenesis, and disruption of IYD dramatically lowered Drosophila fertility and fecundity (Figure 6B). IYD was found to be crucial to sperm development, and suppression by RNAi disrupted the elongation of spermatocytes and blocked sperm formation. The average number of progeny in eya-GAL4/UAS-dmIYD-RNAi resulted in 7 sterile Drosophila samples, whereas the control Drosophila testes, eya-GAL4 and UAS-dmlYD-RNAI resulted in no sterile

Drosophila (Figure 6B). The additional promoters evaluated, nos, bam, c587, and tj did not affect fertility and fecundity in drosophilia.

Iodotyrosine may generate as a signal during maturation, and thus IYD may be responsible for turning the signal off. These RNAi data provide an indication that insects may use iodination/deiodination of tyrosine for regulating certain developmental processes. Example 6: Late stage development is sensitive to IYD

Spermatogenesis in drosophila begins with mitotic division (alternatively, spermatogonia), of germ cells to somatic cells (Figure 7A). Before the transition to meiotic division, the expression of dmIYD (drosophilalYD) is at its peak. Within meiotic division, a spermatocyte evolves into a round spermatide, at which point developmental abnormalities ensue. Elongated spermatids are generated following the formation of the round spermatids.

After RNAi degradation of IYD mRNA, abnormalities were detected in dosophilia testis (Figure 7B). In control testis, nearly 100% showed no phenotype, whereas in IYD RNAi samples, over 80% of the testis showed an abnormal phenotype.

Using phase contrast images of drosophila testis, a considerable difference in control samples versus RNAi knockdown of IYD samples was highlighted (Figure 7C). In control samples, a sperm reservoir was developed, whereas in the RNAi sample, no sperm reservoir was developed. The reservoir serves to ensure successful fertilization by providing the appropriate number of sperm in in the appropriate physiological state for fertilization.

Development of the sperm reservoir is distinctive of late stage development. No sperm reservoir was developed in IYD RNAi drosophilatestis, indicating that suppression of dmIYD disrupted late stage spermatogenesis.

Example 7: Strong expression of isoforms A and B of CG6279 in late stages of

spermatogenesis.

Two isoforms have been reported for Drosophila IYD (dmIYD) in the NCBI databases. These two are related by alternative splicing of the CG6279 gene. Isoform B resembles IYD orthologs from other organisms and includes a membrane domain, an intermediate domain and a catalytic domain (Phatarphekar, A. et a1, Mol. Biosys. 2014, 10, 86-92 and Friedman, J. E. et al. J. Biol. Chem. 2006, 281, 2812-2819). Isoform A is unique to the Drosophila genus by the added presence of a large N-terminal domain (470 amino acids) that does not coincide with any known protein domains listed in the conserved domain database (CDD).

In situ hybridization experiments were conducted with male Drosophila testes, using sequence specific probes (riboprobes) targeting distinct regions of isoform A and B of CG6279 to identify the mRNA expression pattern as well as the stage of mRNA expression during spermatogenesis. In situ hybridization revealed strong expression during the later stages of spermatogenesis, and both riboprobes revealed similar expression patterns (Figures 9A, Figure 9B, Figure 9C, and Figure 9D).

Example 8: Binding affinities of dmIYD Isoform B using various dehalogenation ligands.

Isoform B of the drosophila enzyme (dmIYD) has been cloned, expressed and purified to homogeneity following standard protocols (Phatarphekar, A. et al, Mol. Biosys. 2014, 10, 86-92). This enzyme displayed similar binding affinities for iodo-, diiodo-, bromo- and chlorotyrosine (I-Tyr, I ₂ -Tyr, Br-Tyr and C1-Tyr, respectively), but a 30-fold lower affinity for fluorotyrosine (F-Tyr), as shown in Table 3. Error represents the standard deviation from an average of 3 independent experiments.

Table 3: Dissociation constants (K _D ) for halotyrosines with dmIYD

The enzyme also catalytically dehalogenated all the halotyrosine ligands but F-Tyr. The catalytic efficiency ( k _cat /K _m ) for deiodination and debromination was similar and both were 3-4 fold more efficient than dechlorination, as shown in Table 4. (*) indicated I ₂ -Tyr dehalogenation measured by [ ¹²⁵ I]-iodide released from [ ¹²⁵ I]-I ₂ -Tyr.

Table 4: Steady-state kinetics of dehalogenation catalyzed by dmIYD

Substrate turnover measured by production of tyrosine using HPLC separation and detection Catalytic deiodination of halotyrosines were measured by detecting and quantifying the tyrosine produced in presence of enzyme using reverse phase HPLC. The buffer used for reaction is the same as that used for the [ ¹²⁵ I]-release assay as well as for equilibrium binding measurements (100 niM potassium phosphate, 200 niM KC1 at pH 7.4). Each reaction (final volume of 1 ml) was initiated be addition of 0.5% dithionite (100 μ1 of 5% dithionite in 5% aqueous sodium bicarbonate) rather than the 1% dithionite used for the [ ¹²⁵ I]-release assay. This change minimized interference between tyrosine and dithionite during HPLC separation. The total enzyme used per reaction was 0.08 μΜ and the duration of the reaction was 15-20 mins. M-cresol (30 μΜ) was added to each reaction tube as the internal standard (IS). The reaction was quenched with formic acid (5% final concentration) and the entire reaction mixture was injected manually onto a Microsorb MV 300-5 C18 column analytical HPLC column (Agilent) protected with a manually packed C18 silica guard column. The HPLC method used for analysis is provided in Figure 10.

The concentration of tyrosine produced by IYD was be determined from a standard curve in which known concentrations of tyrosine were spiked into reaction mixtures not containing the halotyrosine but otherwise identical. The ratio of the areas of tyrosine peak to the internal standard peak was plotted against the concentration of tyrosine spiked into the reaction and the best fit line was determined by linear regression. This equation of the line was used to determine the concentration of tyrosine produced in μΜ from a ratio of peak areas of tyrosine to the internal standard.

Example 9: Site-directed mutagenesis on dmIYD identified active site amino acid residues critical for catalysis

Site-directed mutagenesis studies on dmIYD were conducted to identify active site amino acid residues that are critical for catalysis. The E154Q mutation was most detrimental to catalytic efficiency of dmIYD, as shown in Table 5 and may provide a functional knockout mutation in continuing efforts to define the full role of dmIYD in vivo. Kinetic constants were determined by [ ¹²⁵ I]-iodide released from [ ¹²⁵ I]-l ₂ -Tyr.

Table 5: Steady-state kinetics for dehalogenation catalyzed by active site mutants of dmIYD.

Example 10: The significance of the CG6279 for spermatogenesis is confirmed using CRISPER-CAS9 and site-directed mutation studies

Genetic experiments are conducted using CRISPR-CAS9 technology to remove the entire CG6279 gene from the Drosophila genome. Additionally, a loss of function mutation with an E154Q mutation at the genomic level is generated. These experiments confirm the significance of the CG6279 gene for spermatogenesis.

Example 11: Feeding studies using halotyrosines are performed to determine their effects on fertility and spermatogenesis

Feeding studies are performed using halotyrosines added to the standard food for drosophila to determine their effects on fertility and spermatogenesis. This determines whether or not dietary supplements mimic the effect of inhibiting IYD since both allow an unnatural high concentration of halotyrosine to accumulate in vivo.

Example 12: Use of small molecule inhibitors in feeding traps to inhibit reproduction of insects

The feasibility of controlling insect (e.g., mosquito) population by an inhibitor of IYD is assessed through a combination of chemistry, biochemistry and biology. The most efficient inhibitor of IYD requires a 3-step synthesis that is available from the literature. This compound is a pyridone analog of iodotyrosine and previously designed as a mimic of an intermediate formed during enzyme catalysis. Its high affinity was originally measured with porcine IYD. However, the active site structure and catalytic mechanism of all IYDs are similar enough that this inhibitor is effective against IYD from insects. This compound along with others available commercially is used for inhibition studies in vitro and in vivo.

The sequence of IYD from Anopheles gambiae is available, but has not yet been studied. Data assembled from other organisms strongly predict its ability to catalyze deiodination of I _n -Tyr (n= 1,2). The gene is synthesized commercially, expressed heterologously and characterized for its catalytic specificity. Additionally, the enzyme's sensitivity to the known inhibitors, nitrotyrosines and the pyridone derivative, is measured in vitro to confirm their potency before use as feeding supplements.

Feeding studies to measure the effectiveness of simple inhibitors to disrupt fertility

The ability to control mosquito fertility in the field is expedited by easy access to inhibitors of IYD already described in the literature. Nitro- and dinitrotyrosine were identified decades ago as potent inhibitors of IYD in vitro, and more recently a pyridone analog of iodotyrosine was found to maximize affinity to IYD based on the mechanism of catalysis common to all orthologs. These compounds are purchased and synthesized respectively for supplementing mosquito food and are even be tested by direct injection into the body cavity if necessary. Measuring a dose-dependent response establishes the minimum concentration necessary to observe the diminished fertility and fecundity. Since suppression of IYD and the presumed buildup of I- and I ₂ -Tyr are detrimental to Drosophila, mosquitos are also fed with media containing increasing amounts of I- and I ₂ -Tyr in an attempt to overwhelm the catalytic capacity of the native IYD. These treatments recapitulate the expected results of the RNAi investigation.

The most cost effective supplement that suppresses sperm development is then tested in the field. If disruption of I- and I ₂ -Tyr levels is a basis for the sterility, then inhibiting the initial formation of I and I ₂ -Tyr also block spermatogenesis. Biosynthesis of such halogenated amino acids is typically promoted by a peroxidase. Consequently, peroxidase inhibitors such as thiourea, methimazole and propylthiouracil are tested as dietary supplements to inhibit fertility as well. Already, methimazole and propylthiouracil are used as safe and effective drugs for controlling hyperthyroidism. They act by inhibiting the iodination step of thyroxine biosynthesis. Utilizing commercially available female mosquito traps (e.g., In2Care®), an agent that reduces activity or expression of IYD is added to the media to inhibit reproduction of subsequent generations of mosquitos. Larvae eat much more than adults, so spiking a trap like those of "In2Care®" will reach the larvae. Male mosquitoes live on sugar water (e.g., rotting fruit), so in some cases, an agent that reduces activity or expression of IYD is added to a liquid composition to inhibit the reproduction of male mosquitoes.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.