Methods and compositions for non-lethal biological sampling and sex determination in insect larvae

Abstract

Methods and compositions for non-lethal biological sampling of insect larvae are described herein, as are methods and compositions for determining the sex of an insect larvae.

Description

[0001] F& RRefNo.: 55877-0012WO1

[0002] METHODS AND COMPOSITIONS FOR NON-LETHAL BIOLOGICAL SAMPLING AND SEX DETERMINATION IN INSECT LARVAE CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U. S. C. § 119(e) to U. S. Application No. 63 / 738,414 filed on December 23, 2024, and U. S, Application No. 63 / 845,078 filed on July 16, 2025. These applications are incorporated herein by reference in their entirety.

[0003] FIELD OF THE DISCLOSURE

[0004] This disclosure generally relates to methods and compositions for non-lethal biological sampling and sex determination in insect larvae.

[0005] BACKGROUND

[0006] Insects are the most diverse group of animals. With more than a million described species, they represent more than half of all animal species. Insects have a chitinous exoskeleton, a three-part body (head, thorax, abdomen), six legs, and a pair of antennae. Non-lethal biological sampling from insects, particularly from insect larvae, have prevented advancements in insect breeding programs.

[0007] This disclosure describes methods and compositions for non-lethal biological sampling from insect larvae and subsequent sex determination, lire methods and compositions described herein will allow significant improvements in commercial insect strains via, e.g., targeted breeding programs.

[0008] SUMMARY

[0009] This document describes methods and compositions for non-lethal biological sampling of insect larvae, as well as methods and compositions for determining the sex of an insect larvae.

[0010] In one aspect, methods of non-lethally obtaining nucleic acid from an individual larva of a holometabolous insect are provided. Such methods typically include obtaining a biological sample from the individual larva, wherein the biological sample comprisesF& RRefNo.: 55877-0012WO1

[0011] hemolymph and the obtaining is not lethal to the individual larva; and extracting nucleic acid from the biological sample, thereby non-lethally obtaining nucleic acid from the individual larva of the holometabolous insect.

[0012] In some embodiments, the holometabolous insect is a fly. In some embodiments, the fly is a black soldier fly {Hermetia illucens (L.)).

[0013] In some embodiments, the methods further include anesthetizing the individual larva. Anesthetizing can include, for example, placing the individual larva at a temperature of about -20°C for about 5 minutes.

[0014] In some embodiments, the obtaining is performed under sterile conditions (e.g., cleaning surfaces with a disinfectant). In some embodiments, the obtaining is via a needle biopsy. In some embodiments, the needle is between a 22-gauge needle and a 30-gauge needle. For example, a needle biopsy can include piercing the third segment from the head of the larva on the right, dorsal side with a needle using about 1 / 3 of the needle for about 2 seconds. In some embodiment, the needle is inserted without aspiration.

[0015] In some embodiments, the extracting comprises multiple centrifugations. In some embodiments, the nucleic acid is selected from genomic DNA or RNA.

[0016] In some embodiments, further comprising rearing the individual larva at room temp after obtaining the biological sample. In some embodiments, rearing comprises placing the individual larva in a habitat comprising dry feed; and spraying the dry feed with salt water (e.g,, 3% calcium propionate for fungal inhibition) daily for about 7 days.

[0017] In another aspect, methods of non-le thally determining the sex of an individual larva of a holometabolous insect are provided. Such methods typically include obtaining a biological sample from the individual larva, wherein the biological sample comprises hemolymph and the obtaining is not lethal to the individual larva; extracting nucleic acid from tire biological sample; and amplifying a portion of the nucleic acid associated w ith sex determination, thereby non-lethally determining the sex of the individual larva.

[0018] In some embodiments, the holometabolous insect is a fly. In some embodiments, the fly is a black soldier fly {Hermetic! illucens (L.)).

[0019] In some embodiments, the methods further include anesthetizing the individual larva. Anesthetizing can include holding the individual larva at a temperature of about 4°C to about -20°C for about 1 to about 5 minutes.F& RRefNo.: 55877-0012WO1

[0020] In some embodiments, the obtaining is performed under sterile conditions (e.g., cleaning surfaces with a disinfectant). In some embodiments, the obtaining is via a needle biopsy. In some embodiments, the needle is between a 22-gauge needle and a 30-gauge needle. In some embodiments, a needle biopsy includes piercing the third segment from the head of tire individual larva on the right, dorsal side with a needle using about 1 / 3 of the needle for about 2 seconds. In some embodiment, the needle is inserted without aspiration.

[0021] In some embodiments, the extracting comprises multiple centrifugations. In some embodiments, the nucleic acid is selected from genomic DM A or RNA.

[0022] In some embodiments, further comprising rearing the individual larva at room temp after obtaining the biological sample. In some embodiments, rearing comprises placing the individual larva in a habitat comprising dry feed; and spraying the dry feed with salt water (3%) daily for about 7 days.

[0023] In some embodiments, the amplifying comprises amplifying Sequence 1 (SEQ ID NON) and / or Sequence 2 (SEQ ID NO: 10). In some embodiments, the amplifying comprises amplifying using PCR1 conditions (SEQ ID NOs: 1-4) and / or PCR2 conditions (SEQ ID NOs: 5-8).

[0024] In yet another aspect, articles of manufacture are provided that include oligonucleotides for amplifying Sequence 1 (SEQ ID NON) and / or Sequence 2 (SEQ ID NO: 10). In some embodiments, the oligonucleotides for amplifying Sequence 1 comprise SEQ ID NOs: 1-4 and the oligonucleotides for amplifying Sequence 2 comprise SEQ ID NOs: 5-8.

[0025] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0026] Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as wellF& RRefNo.: 55877-0012WO1

[0027] as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

[0028] The term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.

[0029] Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure.

[0030] DESCRIPTION OF DRAWINGS

[0031] The following drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. Like reference symbols in tire drawings indicate like elements.

[0032] FIG. 1 is an electrophoretic gel showing the separation of amplicons used to dete imine the sex of insect larvae.

[0033] DETAILED DESCRIPTION

[0034] Insects, as members of the phylum Arthropoda, exhibit a highly specialized and segmented body plan that has contributed to their evolutionary success. Their anatomy is characterized by an exoskeleton composed of chitin, which provides both structural support and protection while requiring periodic molting for growth. Their bodies are typically divided into three distinct regions: the head, thorax, and abdomen. Tire head houses sensory organs such as simple eyes, compound eyes, antennae, and mouthparts specialized for feeding; the thorax contains the muscles that control movement, including wings (if present) and six jointed legs; and the abdomen is primarily involved in digestion, excretion, and reproduction. The tracheal system for respiration, which allows direct oxygen diffusion without the need for blood transport, oxygenates insect organs and muscles directly. Insects also have an open circulatory system, where hemolymph bathes internal organs without being confined to vessels. Their nervous system isF& RRefNo.: 55877-0012WO1

[0035] decentralized, composed of a dorsal brain with paired ventral nerve cords and ganglia, allowing rapid reflexive responses.

[0036] Like most animals in the Bilateria monophyletic group, insects exhibit bilateral symmetry, meaning their bodies are mirror images along a central axis. This symmetry has several key implications for their anatomy and physiology. Insects have a segmented body plan with a consistent internal organization that supports their survival and functionality. Their organs are strategically arranged to maximize efficiency while maintaining their characteristic bilateral symmetry. The circulatory system is open, meaning hemolymph (fluid similar to blood in invertebrates) flows freely rather than being contained in vessels. The dorsal vessel, which consists of the heart and aorta, runs along the dorsal side of the body, in the center. The heart is located in the posterior abdomen and pumps hemolymph forward through the aorta, which distributes it toward the head before recirculating. The respiratory’ system relies on a tracheal network to deliver oxygen directly to tissues. Spiracles, external openings along the edge of the thorax and abdomen, allow air into the tracheal sy stem, while tracheae branch into fine tracheoles, which facilitate gas exchange at the cellular level. Ihe nervous system consists of a brain in the head that processes sensory’ input and a ventral nerve cord running along the ventral side of the body, centrally located. Paired ganglia in the thorax and abdomen control movement and reflexes independently of the brain. The central arrangement insect physiology along with the symmetry’ and organ placement of insects ensure paired structures (e.g., spiracles, reproductive organs) are evenly distributed and singular structures are centrally located (e.g., ganglia, vessels). This internal arrangement is a key factor in insect adaptability and was used to begin testing the least invasive needle biopsies to be performed on a live insect to ensure their survival to adulthood.

[0037] Despite an increase in rearing of black soldier fly larvae (BSFL; Hermetia illucens (L. )) for the purpose of producing animal feed ingredients, plant and soil products, and other applications, little is known about their genetics and mating structure under wild or commercial farming conditions. The development of non-lethal processes for studying the genetics of the black soldier fly (BSF) at their immature larval stage enables the development of effective population management strategies to maximize yields and genetic strains in commercial farms.

[0038] A major bottleneck in directing BSF population growth and selection is the inability to determine the sex of the individual insect prior to reaching adulthood, whenF& RRefNo.: 55877-0012WO1

[0039] sexual organs are developed, and the sex is visibly identifiable. To date, the inability to establish sex prior to insect adulthood while maintaining the sampled insects alive has limited the application of genetic studies (e.g., genetics selection, population structure). The methods described herein encompass a non-lethal method of collecting a biological sample (e.g., nucleic acids such as DNA (e.g., genomic DNA) or RNA) from live larvae (i. e., prior to their adult life stage), using that biological sample for genetic studies including, for example, sex determination, and then using the results of those genetic studies to educate breeding strategies.

[0040] Methods

[0041] The methods described herein for non-lethally obtaining a biological sample from individual BSF larvae (BSFL) can be applied to the larvae of virtually any holometabolous insect. Holometabolous insects undergo complete metamorphosis (i.e,, egg. larva, pupa, and adult), and include, without limitation, flies, moths, butterflies, beetles, wasps, ants, and bees. Representative types oflarvae that can be used in the methods described herein include, without limitation, Eruciform (e.g., caterpillar), Scarabaeifonn larvae (e.g., grub), Campodeiform (e.g., antlion), Elateriform (e.g., family Elateridae), and Vermiform (e.g., maggot, black soldier fly larva). Tire methods described herein also can be applicable to hemimetabolous (incomplete metamorphosis) nymphs / naiads. It would be appreciated that the outcome of such methods likely depends on the specific life cycle of the insect species (e.g., instars / developmental stages and the associated size of each), an appropriately sized needle, and whether any larval exoskeleton present is penetrable by tire given needle.

[0042] The particular instar / stage of the larvae used can be an important factor in the outcome of the method. For BSF, the early stadium of the sixth larval instar (27-day old) yielded the highest occurrence of survival through the adult stage, i.e., fly eclosion, however, similar amounts of DNA (ng / pl) were obtained at other stages such as the 4thto 6thinstar larvae.

[0043] Obtaining a biological sample from an individual larva without sacrificing the larva was developed with the goal of being the least invasive and traumatic to the larvae, based on both external and internal physiology. Multiple locations were tested, predominantly focusing on the dorsal side of the larvae. Insects are bilaterally symmetrical, and the internal organs should be distributed in the same way, stemmingF& RRefNo.: 55877-0012WO1

[0044] from the center of the body. We tested locations predominantly on the right side of the larval body, at thoracic segment 3, and abdominal segments 2, 4, 6, and 8. Within the right side of each segment, the needle insertion was centered to avoid the heart tube and trachea running along the center of the insect, and the spiracle on the lateral edge of the segment, lire left side of the body would be expected to work equally well, taking into account similar spatial considerations. From the segments tested, thoracic segment 3 (i.e., third segment from the head) yielded the highest occurrence of survival of larvae through the adult stage, i.e., fly eclosion, but larvae survived through to adulthood when the biological sample was obtained from the abdominal segments. None of the segments tested resulted in total lethality.

[0045] The gauge of needle and the degree or depth of insertion varied slightly by user but, typically, a 22-gauge (G) to 30-gauge (G) needle (e.g., 22G, 23G, 24G, 25G, 26G, 27G, 28G, 29G or 30G) can be used and at least about one-third (e.g,, about 4 mm) and no more than about one-half (e.g., about 7 mm) of the length of the needle insertion is recommended for needle biopsy. As described herein, a needle biopsy on an insect larva generally includes piercing the third segment from the head of the larva on the right, dorsal side with about one-third (e.g., about 4 mm; e.g,, greater than about one-fourth (e.g., about 3 mm) and less than about one-half (e.g., about 7 mm)) of the needle for about 2 seconds (e.g., about 1 second, about 3 seconds, about 1-3 seconds). Typically, the biological sample is obtained when the needle is introduced into the larva and little to no aspiration is performed. Insertion of the needle in the absence of aspiration can minimize damage to the larva and increase the survival of the larva.

[0046] Obtaining a biological sample from an individual larva without sacrificing the larva typically includes obtaining hemolymph using a needle biopsy under sterile conditions (e.g., by disinfecting surfaces and equipment). In addition, the larvae to be sampled can be anesthetized prior to the biopsy by, for example, holding the larvae at about -20°C (e.g., about -25°C to about -15°C, about -22°C to about -18°C) for about 5 mins (e.g., about 3 minutes, about 4 minutes, about 6 minutes, about 7 minutes, about 3-7 minutes, about 4-6 minutes, about 5-7 minutes).

[0047] Extracting nucleic acids from biological samples are known in the art, and extracting nucleic acids from small biological samples also are known in the art.

[0048] Typically, extracting nucleic acids includes multiple centrifugations, one or moreF& RRefNo.: 55877-0012WO1

[0049] precipitations, and one or more washes, all of which are known in the art. Additionally, techniques to preserve the viability of nucleic acids are known in the art.

[0050] After the biological sample is obtained, the larva is reared in a room temperature environment under conditions that ensure survival. As described herein, rearing can include placing the individual larva in a habitat that includes dry feed (see, e.g., Table 1) and spraying the dry’ feed with saltwater (e.g., 3% calcium propionate) at least once daily for about 7 days to inhibit fungal formation. The recovery’ methods using the feed and water were necessary to provide harborage and feed for the final molt to the seventh larval instar, also known as the pre-pupa. Additionally, the calcium propionate water mitigates mold growth otherwise prevalent due to a single larva’s inability’ to consume and process the feed before other microorganisms. After the pre-pupa become juvenile insects (e.g., about 7 days for BSF), the juveniles are allowed to mature into adults using routine methods.

[0051] As described herein, one of the applications of non-lethally obtaining a biological sample from an insect larva is sex determination. Sex determination, as well as other genetic analysis, in the larval stage can significantly improve breeding programs for insects, particularly those with an extremely short life-cycle (e.g,, 14 days or less; 10 days or less; 7 days or less). Although any number of techniques can be used to evaluate the biological sample and the nucleic acids therein, amplification of the nucleic acids in the biological sample using polymerase chain reaction (PCR) is the most common, particularly when the sample or the amount of nucleic acids in the sample are limited. Numerous types of PCR amplification methods are known in the art. See, for example, PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995.

[0052] Genetic loci associated with sex determination in insect larvae are described herein, as are oligonucleotides that amplify such loci. Tlie genetic loci associated with sex determination in insect larvae can be, without limitation, on the Y chromosome, which is present in male BSF. Multiple sets of primers are described herein (PCR1 (SEQ ID NOs: 1-4) and PCR2 (SEQ ID NOs: 5-8)) that produce two amplicons depending on BSF sex. U sing the primers described herein, a single amplicon is produced for females and two different sized amplicons are produced for males. The locus corresponding to PCR2 (SEQ ID NO:s: 5-8) produces the second amplicon associated with the Y chromosome presentF& RRefNo.: 55877-0012WO1

[0053] in the male BSF. It would be understood that other primers could be designed to amplify the same or a different sex-linked locus.

[0054] Many insects, including black soldier flies, have the XY sex-determination system where males are the heterogametic (XY) sex. Therefore, a PCR-based sex determination can amplify at least a region from the Y -chromosome, where an amplicon is produced only if male DNA is present. Insect sex chromosomes have been studied in detail (see, e.g., Vicoso and Bachtrog, 2015, PLoS Biol., 13:el002078), and the genomic sequence of many insects are known, which allows for the identification of Y-chromosome sequences with high mapping qualify for use in the sex determination assay.

[0055] Fortlie sex determination of BSF as described herein. Primer Set 1 was used to amplify a control autosomal region (“Sequence 1”) referred to as Elongation Factor 1 (EFT) (GenBank Accession No. DQ168685.1; SEQ ID NO: 9), while Primer Set 2 was used to amplify’ a sex -linked Y-Chromosome region (“Sequence 2”) to produce an 303 nt amplicon (SEQ ID NO: 10). See Example 5.

[0056] Nucleic Acids

[0057] Nucleic acids can include DNA and RNA and includes nucleic acids that contain one or more nucleotide analogs. A nucleic acid can be single stranded or double stranded, and the strandedness of the nucleic acid will depend upon its intended use.

[0058] One nucleic acid can differ in sequence from another nucleic acid. In calculating percent sequence identity, tw o sequences are aligned and the number of identical matches of nucleotides or amino acid residues betw een the two sequences is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of aligned nucleotides or amino acid residues) and multiplied by 100 to arrive at a percent sequence identify value. It will be appreciated that the length of the aligned region can be a portion of one or both sequences up to the full-length size of the shortest sequence. It also will be appreciated that a single sequence can align with more than one other sequence and hence, can have different percent sequence identity values over each aligned region. It is noted that the percent identity value is usually rounded to the nearest integer. For example, 78.1%, 78.2%, 78.3%, and 78.4% are rounded down to 78%, while 78.5%, 78,6%, 78.7%, 78.8%, and 78.9% are rounded up to 79%. It is also noted that the length of the aligned region is always an integer.F& RRefNo.: 55877-0012WO1

[0059] The alignment of two or more sequences to determine percent sequence identity can be performed using the algorithm described by Altschul et al. (1997, Nucleic Acids Res., 25:3389 3402) as incorporated into BLAST (basic local alignment search tool) programs, available at ncbi.nlm.nih.gov on the World Wide Web. BLAST searches can be performed to determine percent sequence identity between a nucleic acid and any other sequence or portion thereof aligned using the Altschul et al. algorithm. BLASTN is the program used to align and compare the identity between nucleic acid sequences, while BLASTP is the program used to align and compare the identity between amino acid sequences. When utilizing BLAST programs to calculate the percent identity between a sequence and another sequence, the default parameters of the respective programs generally are used.

[0060] As used herein, an “isolated” nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acids that are usually associated with the nucleic acid molecule in the genome. Thus, an “isolated” nucleic acid molecule includes, without limitation, a nucleic acid molecule that is free of sequences that naturally flank one or both ends of the nucleic acid in the genome of the organism from which the isolated nucleic acid molecule is derived (e.g., a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease digestion). In addition, an isolated nucleic acid molecule can include an engineered nucleic acid molecule such as a recombinant or a synthetic nucleic acid molecule, A nucleic acid molecule existing among hundreds to millions of other nucleic acids within, for example, a nucleic acid library (e.g., a cDNA, or genomic library) or a portion of a gel (e.g., agarose or polyacrylamine) containing restriction- digested genomic DNA typically is not considered an isolated nucleic acid.

[0061] Isolated nucleic acids can be obtained using techniques known in the art. For example, isolated nucleic acids can be obtained using, without limitation, recombinant nucleic acid technology, and / or the polymerase chain reaction (PCR). General PCR techniques are described, for example in PCR Primer: A Laboratory’ Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory' Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate a nucleic acid. Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides. In addition, isolated nucleic acids can be obtained by mutagenesis. For example, an isolated nucleic acid that shares identity with an art known sequence can be mutated using commonF& RRefNo.: 55877-0012WO1

[0062] molecular cloning techniques (e.g., site-directed mutagenesis). Possible mutations include, without limitation, deletions, insertions, substitutions, and combinations thereof.

[0063] Vectors containing nucleic acids also are provided. Vectors, including expression vectors, suitable for use in the methods described herein are commercially available and / or produced by recombinant DNA technology methods routine in the art. A vector containing a nucleic acid can have elements necessary for expression operably linked to such a nucleic acid, and further can include sequences such as those encoding a selectable marker (e.g., an antibiotic resistance gene), an origin of replication, and / or those that can be used in purification of a polypeptide (e.g., 6xHis tag).

[0064] Elements necessary’ for expression include nucleic acid sequences that direct and regulate expression of nucleic acid coding sequences. Elements necessary for expression can include a promoter sequence, introns, enhancer sequences, response elements, or inducible elements that modulate expression of a nucleic acid. Elements necessary for expression can be of bacterial, yeast, insect, mammalian, or viral origin and vectors can contain a combination of elements from different origins. Elements necessary for expression are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology, 185, Academic Press, San Diego, CA, As used herein, operably linked means that a promoter and / or other regulatory element(s) are positioned in a vector relative to a nucleic acid in such a way as to direct or regulate expression of the nucleic acid. Many methods for introducing nucleic acids into host cells, both in vivo and in vitro, are known to those skilled in the art and include, without limitation, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral -mediated nucleic acid transfer.

[0065] Another aspect pertains to host cells into which a vector, e.g., an expression vector, or an isolated nucleic acid molecule has been introduced. The term “host cell” refers not only to tire particular cell but also to the progeny or potential progeny of such a cell. A host cell can be any prokaryotic or eukaryotic cell. For example, nucleic acids can be expressed in bacterial cells such as E. coli, or in insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.F& RRefNo.: 55877-0012WO1

[0066] Embodiments

[0067] Embodiment 1 is a method of non-lethally obtaining nucleic acid from an individual larva of a holometabolous insect, comprising: obtaining a biological sample from the individual larva, wherein the biological sample comprises hemolymph and the obtaining is not lethal to the individual larva; and extracting nucleic acid from the biological sample, thereby non-lethally obtaining nucleic acid from the individual larva of the holometabolous insect.

[0068] Embodiment 2 is the method of Embodiment 1, wherein the holometabolous insect is a fly.

[0069] Embodiment 3 is the method of Embodiment 2, wherein the fly is a black soldier fly (Hermetia illucens (L.)).

[0070] Embodiment 4 is the method of any one of Embodiments 1 to 3, wherein the obtaining is performed under sterile conditions.

[0071] Embodiment 5 is the method of Embodiment 4, wherein the sterile conditions comprise cleaning surfaces with a disinfectant.

[0072] Embodiment 6 is the method of any one of the preceding Embodiments, further comprising anesthetizing the individual larva.

[0073] Embodiment 7 is the method of Embodiment 6, wherein the anesthetizing comprises placing the individual larva at a temperature of about -20°C for about 5 minutes.

[0074] Embodiment 8 is the method of any one of the preceding Embodiments, -wherein the obtaining is via a needle biopsy.

[0075] Embodiment 9 is the method of any one of the preceding Embodiments, wherein the obtaining the biological sample non-lethally comprises piercing the third segment from the head of the larva on the right, dorsal side with a needle.

[0076] Embodiment 10 is the method of Embodiment 9, wherein the piercing comprises inserting about 1 / 3 of the needle for about 2 seconds.

[0077] Embodiment 11 is the method of any one of the preceding Embodiments, wherein the needle is between a 22-gauge needle and a 30-gauge needle.

[0078] Embodiment 12 is the method of any one of the preceding Embodiments, wherein the extracting comprises multiple centrifugations.

[0079] Embodiment 13 is tire method of any one of the preceding Embodiments, wherein the nucleic acid is selected from genomic DNA or RNA.F& RRefNo.: 55877-0012WO1

[0080] Embodiment 14 is the method of any one of the preceding Embodiments, further comprising rearing the individual larva at room temp after obtaining the biological sample.

[0081] Embodiment 15 is the method of Embodiment 14, wherein rearing comprises placing the individual larva in a habitat comprising dry feed; and spraying the dry feed with salt water once daily for about 7 days.

[0082] Embodiment 16 is a method of non-lethally determining the sex of an individual larva of a holometabolous insect, comprising: obtaining a biological sample from the individual larva, wherein the biological sample comprises hemolymph and the obtaining is not lethal to the individual larva; extracting nucleic acid from the biological sample; and amplifying a portion of the nucleic acid associated with sex determination, thereby non-lethally determining the sex of the individual larva.

[0083] Embodiment 17 is the method of Embodiment 16, wherein the holometabolous insect is a fly.

[0084] Embodiment 18 is the method of Embodiment 17, wherein the fly is a black soldier fly (Hermetia illucens (L.)).

[0085] Embodiment 19 is the method of any one of Embodiments 16 to 18, wherein the obtaining is performed under sterile conditions.

[0086] Embodiment 20 is the method of Embodiment 19, wherein the sterile conditions comprise cleaning surfaces with a disinfectant.

[0087] Embodiment 21 is the method of any one of Embodiments 16 to 20, further comprising anesthetizing the individual larva.

[0088] Embodiment 22 is the method of Embodiment 21, wherein the anesthetizing comprises holding the individual larva at a temperature of about -20°C for about 5 minutes.

[0089] Embodiment 23 is the method of any one of Embodiments 16 to 22, w herein the obtaining is via a needle biopsy.

[0090] Embodiment 24 is the method of any one of Embodiments 16 to 23, wherein obtaining the biological sample non-lethally comprises piercing the third segment from the head of the individual larva on the right, dorsal side with a needle.

[0091] Embodiment 25 is the method of Embodiment 24, wherein the piercing comprises about 1 / 3 of the needle for about 2 seconds.F& RRefNo.: 55877-0012WO1

[0092] Embodiment 26 is the method of any one of the preceding Embodiments, wherein the needle is between a 22-gauge needle and a 30-gauge needle.

[0093] Embodiment 27 is the method of any one of Embodiments 16 to 26, -wherein the extracting comprises multiple centrifugations.

[0094] Embodiment 28 is the method of any one of Embodiments 16 to 27, wherein the nucleic acid is selected from genomic DNA or RNA.

[0095] Embodiment 29 is the method of any one of Embodiments 16 to 28, further comprising rearing the individual larva at room temp after obtaining the biological sample.

[0096] Embodiment 30 is the method of Embodiment 29, wherein rearing comprises placing the individual larva in a habitat comprising dry feed; and spraying the dry feed with salt water (3%) daily for about 7 days.

[0097] Embodiment 31 is the method of any one of Embodiments 16 to 30, wherein the amplifying comprises amplifying SEQ ID NO:9 and / or SEQ ID NO: 10.

[0098] Embodiment 32 is an article of manufacture, comprising oligonucleotides for amplifying Sequence 1 (SEQ ID NO:9) and / or Sequence 2 (SEQ ID NO: 10).

[0099] Embodiment 33 is the article of manufacture of Embodiment 32, wherein the oligonucleotides for amplifying Sequence 1 comprise SEQ ID NOs: 1-4 and the oligonucleotides for amplifying Sequence 2 comprise SEQ ID NOs: 5-8.

[0100] In accordance -with the present invention, there may be employed molecular biology, microbiology, biochemical, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. The invention will be further described in the following examples, which do not limit the scope of the methods and compositions of matter described in the claims.

[0101] EXAMPLES

[0102] Example 1 — Hemolymph Needle Biopsy DNA Extraction

[0103] Black soldier fly larvae (BSFL; Hermetia illucens (L.)) individuals were obtained from populations maintained year-round in the Genetics R& D laboratory, EnviroFlight, Apex, North Carolina. All BSFL, were housed in containers proven suitable for their harborage and feeding. The populations have been maintained at 27°C and 55% relativeF& RRefNo.: 55877-0012WO1

[0104] humidity. Individual larvae at 23 days of age were collected for hemolymph needle biopsy, DNA extraction, and individual BSFL rearing to adulthood.

[0105] The working surface was disinfected using a bleach cleaner before starting the hemolymph needle biopsy and sterile technique practices used to prevent contamination of all samples and surfaces. An individual BSFL was collected and placed in cooler at 4 °C for 5 minutes to anesthetize the BSFL prior to the biopsy. Immediately after removing the BSFL from the cooler, a single-use needle (26 G ’ / ?) was used to collect a hemolymph sample. Holding the needle at an angle, the body of the BSFL was pierced at the third segment from the head on the right, dorsal side. One-third of the needle length was inserted and held for 2 seconds along the length of the body to avoid damage to the BSFL. The needle was placed into a microcentrifuge tube, needle-end first, and closed. The BSFL was placed aside in a separate microcentrifuge tube and held for individual rearing preparation an hour after the needle biopsy. The BSFL tube is kept open and held at room temperature.

[0106] In the needle tube, 25 uL of QuickExtract™ (QE) DNA Extraction Solution (Lucigen) was added, placing the QE directly into the hub of the needles, as close to the center as possible, and the tube closed. The needle tube was placed in a microcentrifuge and spun for 30 seconds at 3000 rpm to expel the hemolymph and QEB from the needle and into the base of the tube. With the needle still in the tube, the needle tube was vortexed the tubes for 3 seconds and spun for 30 seconds at 3000 rpm. The needle tube was then placed in a heat block for incubation at 65 °C for 40 minutes. The needle tube was removed from incubation and vortexed for 15 seconds then spun for 30 seconds at 3000 rpm. The needle tube was then placed in a heat block for incubation at 98°C for 10 minutes. The needle tube was removed from incubation, allowed to cool briefly for 5 minutes, then spun for 30 seconds at 3000 rpm. The needle was then carefully removed from the needle tube using sterile forceps and discarded properly. The tube with the remaining hemolymph DNA sample was then placed in a freezer at -20°C for future use in sex determination polymerase chain reaction (PCR). DNA concentration and purity were analyzed on a Qubit Fluorometer.

[0107] Example 2 — Individual BSFL Rearing

[0108] The working surface was disinfected using a bleach cleaner before starting the individual rearing placements and sterile technique practices used to preventF& RRefNo.: 55877-0012WO1

[0109] contamination of all samples and surfaces. A condiment cup and condiment cup lid with a 5 / 16 drill bit hole were prepared to house the individual BSFL. A pre-mixed dry feed was prepared (see Table 1) and teaspoon was added to the condiment cup. A 3% saltwater solution was prepared using calcium propionate and placed in a spray bottle with a direct stream, approximately 2 mL per 1 direct stream spray. The feed in the cup was then sprayed with 1 direct stream of the saltwater solution to complete the habitat cup set up, The biopsied BSFL was placed inside the habitat cup and closed with cup lid. The BSFL was observed daily and sprayed 1 direct stream of water into the habitat cup for approximately 7 days. More feed was added to the habitat cup if the BSFL had not reached the pre-pupal stage and the feed was almost gone. Upon reaching the pre-pupal stage, watering was stopped and the cup was placed aside and a piece of tape was place on half of the lid hole to allow the insect to complete their metamorphosis to adult fly. The insect was observed daily until the adult fly emerged. On the day of the fly emergence, the BSF was carefully taken out of the cup and sex identified using a compound microscope to examine the external sexual organs. Male BSF are characterized by a pair of hooks located at the last segment of the abdomen. Female BSF are characteri zed by a telescoping ovipositor with two cerci located at the last segment of the abdomen.

[0110] Table 1

[0111] Ingredients

[0112] Distillers dried grains 49.1

[0113] Bakery by-product meal 49.0

[0114] Calcium carbonate 1.0

[0115]

[0116] Calcium chloride 0.85

[0117] Example 3 — Polymerase Chain Reaction and Gel Electrophoresis

[0118] The BSFL Hemolymph DNA sample was taken from storage and thawed to begin downstream amplification. Two sets of primers for two PCRs were designed to determine BSFL sex based on successful amplification and PCR product size visualization via gel electrophoresis. The primers used were as follows:

[0119] PCR 1

[0120] Primer Set 1-1, Product Size 866 bp

[0121] Forward Primer 5 ’-TGG TAC GTC ACA AGC TGA TTG CGC CGT-3’ (SEQ ID NO:1)F& RRefNo.: 55877-0012WO1

[0122] Reverse Primer 5’-CCT TGG GGT TCT CCT CGG TGG TCT T-3’ (SEQ ID NO: 2)

[0123] Primer Set 1-2, Product Size 429 bp

[0124] Forward Primer 5 ’-ATT CGT TCT GAG GAC TGT AGT ACC GAT C-3’ (SEQ ID NO:3)

[0125] Reverse Primer 5 ’-GGT ATC TGG AAC AA T ATA CIG AGT AGT TAA TGT GIT C-3’ (SEQ ID NON)

[0126] PCR 2

[0127] Primer Set 2-1, Product Size 678 bp

[0128] Forward Primer 5’-GCT GGT ATC TCA AAG AAC GGA CAA ACC C- 3’ (SEQ ID N0:5)

[0129] Reverse Primer 5’ -GGG A EG GTT GAG CAC GAT GAC TIG G-3’ (SEQ ID NO: 6)

[0130] Primer Set 2-2, Product Size 303 bp

[0131] Forward Primer 5 ’-GGT CGC CTT TGG GA A GTC TAC AC A A-3’ (SEQ ID NO: 7)

[0132] Reverse Primer 5 ’-CCT TCA GAT ATT TGG TAA GTT GAA TAA TTT GTT GGG GG-3’ (SEQ ID NO:8)

[0133] PCR 1 primers were used to amplify genomic DNA in a 20 pL reaction mixture containing tire following: 2 pL genomic DNA, 10 pL Q5 2x Master Mix, 1 pL of 0.5 pM of each forward and reverse primer, and 7 pL ultrapure water. To assure validity of the data, both a negative control (reaction mixture with water instead of any DNA sample) and two positive controls (a DNA sample from an adult male BSF and a DNA sample from an adult female BSF) were incorporated for primer-pair testing. Amplification of DNA with Primer Set 1 was completed in a 96 well plate using the following thermal profile: initial denaturation at 98°C for 5 minutes followed by 20 cycles of: denaturation at 98°C for 60 seconds, 64°C for 45 seconds, and extension at 72°C for 30 seconds, with a final extension of 72°C for 5 minutes.

[0134] PCR 2 primers were used to amplify’ the amplicon from PCR 1 in a 20 pL reaction mixture containing the following: 2 pL PCR 1 template, 10 pL Q5 2x Master Mix, I pL of 0.5 pM of each forward and reverse primer, and 7 pL ultrapure water. To assure validity of the data, both a negative control (reaction mixture with water instead of any DNA sample) and two positive controls (a DNA sample from an adult male BSF and a DNA sample from an adult female BSF) were incorporated for primer-pair testing.

[0135] Amplification of DNA with Primer Set 2 was completed in a 96 well plate using the following thermal profile: initial denaturation at 98°C for 5 minutes followed by 35 cycles of denaturation at 98°C for 60 seconds, 64°C for 45 seconds, and extension at 72°C for 30 seconds, with a final extension of 72°C for 5 minutes.F& RRefNo.: 55877-0012WO1

[0136] The amplified DNA products were separated by size using on 2% agarose gel electrophoresis and then stained with GelRed Nucleic Acid Stain. The amplified fragments were visualized and the sex of the BSFL was determined based on the number of fragments produced by the amplification, with a single band (i.e., corresponding to a fragment of 678 bp) representing a female BSFL and two bands (i.e., corresponding to one fragment at 678 bp and one fragment at 303 bp) representing a male BSFL. The sex determination was confirmed and validated when the adult fly emerged.

[0137] Example 4 — BSFL DNA Extraction with Sex Determination and Adult Fly Validation A random sample of BSFL at 23 days of age were collected, DNA was extracted using the hemolymph needle biopsy described herein, and each individual BSFL was subsequently reared. PCR and gel electrophoresis was conducted to identify the BSFL sex and confirmed later by observing the sex organ of the adult fly. FIG. 1 illustrates the PCR amplification of BSFL samples (lanes 1-8), Female BSF DNA positive control (lane 9), Male BSF DNA positive control (lane 10), and no-template negative control (lane 11). Table 2 shows the sex determination using BSFL DNA and PCR (i.e., from FIG. 1) and visual inspection once the same BSFL emerged as an adult. Tire correct sex of the BSFL samples was determined using PCR 100% of the time prior to reaching adulthood.

[0138] Additionally, 100% of the BSFL were kept alive during and after tire needle biopsy and could be used for selected mating experiments as adults.

[0139] Table 2

[0140] Sex determination Sex determination Sample Sample Name (PCR on living (visual inspection larvae) of adult Fly) Marker 10000 bp DNA Ladder

[0141] 1 BSFL Sample 1 M M

[0142] 2 BSFL Sample 2 F F

[0143] 3 BSFL Sample 3 F F

[0144] 4 BSFL Sample 4 F F

[0145] 5 BSFL Sample 5 F F

[0146] 6 BSFL Sample 6 F F

[0147] 7 BSFL Sample 7 M M

[0148] 8 BSFL Sample 8 M M

[0149] 9 Female BSF F F

[0150] 10 Male BSF M M

[0151]

[0152] 11 No template none noneF& RRefNo.: 55877-0012WO1

[0153] Example 5 - Sequences

[0154] Accession DQ168685 is the target region (“Sequence 1”, SEQ ID NO: 9) for Primer Set 1.

[0155] ctgtggaagt tcgaaacctc caagtac tac gtgaccatca tcgatgcccc tggacatcgt gatttcatca agaacatga tactggtacg tcacaagctg attgcgccgt gttgattgtt gccgccggta ccggtgaatt cgaagctggt atctcaaaga acggacaaac ccgtgaacac gccttgttgg ccttcacttt gggtgtcaag caattgattg tcggcatcaa caagatggac tcctccgaac caccatacag cgaggcccgt tatgaggaaa tcaagaagga agtctagtcg tacatcaaga agatcggtta caatccagcc gctgttccat tcgtcccaat ctccggatgg cacggagaca acatgttgga accatcaacc aacatgaact ggttcaaggg atgggccatc gaacgcaagg aaggcaaggc tgaaggtaag accttgatcg atgctttgga cgctatcttg ccacccaccc gtcaaaccga gaagcctatt cgtctcccac tccaggacgt gtacaaaatt ggcggtatcg gaacagtacc agtaggtcga gttgaaactg gtgtcttgaa accaggtatg gttgtcgtct tcgccccagc caacatcacc actgaagtma agtccgtyga aatgcaccac gaagctttgc ccgaggccgt gcccggtgac aacgttggtt tcaacgttaa gaacgtctcc gtcaaggaat tgcgtcgtgg ctacgttgct ggygactcca agagcagccc accccgcgga gctgctgact tcaacgccca agtca cgtg ctcaaccatc ccggtcaaat ctagaacggy tacaccccag tgttggattg ycaaacwgcy cacatwgcct gcaaattcta cgaaatcaag gagaaggtcg atcgtcgttc cggcaagacc accgaggaga accccaaggc catcaagtct ggtgatgccg ccatcgtcat cctcgtccca accaaaccct tgtgcgtcga atcgttccag gagttccctc cattgggtcc yttcgctgta cgtgaaat

[0156] Accession NW 024037034 (region 19463-21289) is the target region (“Sequence 2”, SEQ ID NO: 10) for Primer Set 2.

[0157] atgcgttctg agagctgtgg taccggtcaa aaggtcgcct ttgcaaagtc tacacaagca gtttgtggtc attattaccc aagtgtttct attcataaat ttacttaacg gataaag ca attgaattca ccggaatcag tcaccctgcg ttatccttcc ccaactt caa aagtactcaa atgaaaattg cttttgctcc gat tctacc gcttgttgca ttgtatccat caaatatgcg aacgttgttg gctcaagaat tcctccaaca aattattcac cttaccaaat at ctgaagct aaattggtga ccgaggaacg aacttctttt aagaaatcga ttgagttcaa gtcttggagc acattaacta ctcagtatgt tgttccagat attaattacc tttacttctt ccaattagta gctccctcca cctccctcag caacatgaga accacatcac tatgtcagat tagtcaaatg aaat ggga taatctccat tcagtcgacg gcacctactg catctcactc gacattcctc tcaacaacga cggagtcgat t c c a a a c c a gttctgagtt cgacaacgat tataagtgtc ccatttgcaa ggaactctcc actaacgcag tcaaaggaaa atccctgtgg aagactatcc tcacaatata attggacgtg tcccgtatat aaagtaaaga aaagcacatc agattccccg gcacattcca t tcaaccaca aacttctgta tacgactttc taactaccct acaaggaaca ggacgatctt tcactccgct ccagatcatg gtatattctc cattcttttc gatgagtagt gtggcaggtt acacaacaga cgtttggcgc aatcattttg acagttatct caaaattcaa gtatcaggta ggacgtcgag ccaaaacttc cgaagtctga ctaaccgacc ttatcctgag agatatcaag gtttttccat tttgatattt actgaaaaga aacggtattt ggaaagcatt aacactccct tcaatattat atgcaaggaa tatccaagca ttattcgcac tataacagat ccgaaaagcc gttcatcttg caaaacgttg gagcgcaatt at ccgacaga atggatatgc acttcacgtc aaacactgcc gtggaataca acgcggaaga catggagttt gcattccctt tactagaatt ttggatatca gacaggaaaa gtcataacat tttcaaaggt gtaccaagct gaaacacaaa cttaggctta tacacacctg acagtcgagc agctaggggg ataatcttca acacccttcc ggaatgcaat atactctgca ctatgttccg tgagtaggtt ggtgggatat agaat agag t c c a i t ar tcgttctgag gactgtagta ccgatcaaag ggtcgccttt gggaagtcta cacaagcagt ttgcagtcat tattacccaa gtgattctat ucataa utg ac caaaaga aaataatttc gttgtggatt aataggtctt catggattaa gtccatcgaa tccaccggaa tcagtcgttc ggcgttatcc t tccccgcct tcaaaagccc tcaaataaaa attccttttg ctccgatttc taccgctttg tgcactttat ccatgaaata tgcgaacgtt at uggctcaa gaattccccc a a c a a a 11 a t tcaacttacc a atatctg aggtaaa cg atgactgaga aacgaacttc ttttcagaaa tcgattgaaa tcaattcttg gaacac uta actaatcagt atattgttcc agataccF& RRefNo.: 55877-0012WO1

[0158] It is to be understood that, while the methods and compositions of matter have been described herein in conjunction with a number of different aspects, the foregoing description of the various aspects is intended to illustrate and not limit the scope of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims.

[0159] Disclosed are methods and compositions that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are disclosed. That is, while specific reference to each various individual and collective combinations and permutations of these compositions and methods may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular composi tion of matter or a particular method is disclosed and discussed and a number of compositions or methods are discussed, each and every combination and permutation of the compositions and the methods are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.

Claims

F& RRefNo.: 55877-0012WO1WHAT IS CLAIMED IS:

1. A method of non-lethally obtaining nucleic acid from an individual larva of a holometabolous insect, comprising:obtaining a biological sample from the individual larva, wherein the biological sample comprises hemolymph and the obtaining is not lethal to the individual larva; andextracting nucleic acid from the biological sample,thereby non-lethally obtaining nucleic acid from the individual larva of the holometabolous insect,2. The method of claim 1, wherein the holometabolous insect is a fly.

3. The method of claim 2, wherein the fly is a black soldier fly (Hermetia illucens (L.)).

4. The method of claim 1, wherein the obtaining is performed under sterile conditions.

5. The method of claim 4, wherein the sterile conditions comprise cleaning surfaces with a disinfectant.

6. The method of claim 1, further comprising anesthetizing the individual larva.

7. The method of claim 6, wherein the anesthetizing comprises placing the individual larva at a temperature of about -20°C for about 5 minutes,8. The method of claim 1, wherein the obtaining is via a needle biopsy.

9. The method of claim 1, wherein the obtaining the biological sample non-lethally comprises piercing the third segment from the head of the larva on the right, dorsal side with a needle.F& RRefNo.: 55877-0012WO110. The method of claim 9, wherein the piercing comprises inserting about 1 / 3 of the needle for about 2 seconds.

11. The method of claim 9, wherein the needle is between a 22-gauge needle and a 30-gauge needle.

12. The method of claim 1, wherein the extracting comprises multiple centrifugations.

13. The method of claim 1, wherein the nucleic acid is selected from genomic DNA or RNA.

14. The method of claim 1, further comprising rearing the individual larva at room temp after obtaining the biological sample.

15. The method of claim 14, wherein rearing comprises placing the individual larva in a habitat comprising dry feed; and spraying the dry feed with salt water once daily for about 7 days.

16. A method of non-lethally determining the sex of an individual larva of a holometabolous insect, comprising:obtaining a biological sample from the individual larva, wherein the biological sample comprises hemolymph and the obtaining is not lethal to the individual larva:extracting nucleic acid from the biological sample; andamplifying a portion of the nucleic acid associated with sex determination, thereby non-lethally determining the sex of the individual larva.

17. The method of claim 16, wherein the holometabolous insect is a fly.

18. The method of claim 17, wherein the fly is a black soldier fly (Hermetia illucens (L.)).F& RRefNo.: 55877-0012WO119. The method of claim 16, wherein the obtaining is performed under sterile conditions.

20. The method of claim 19, wherein the sterile conditions comprise cleaning surfaces with a disinfectant.

21. The method of claim 16, further comprising anesthetizing the individual larva.

22. The method of claim 21, wherein the anesthetizing comprises holding the individual larva at a temperature of about -20°C for about 5 minutes.

23. The method of claim 16, wherein the obtaining is via a needle biopsy.

24. The method of claim 16, wherein obtaining the biological sample non-lethally comprises piercing the third segment from the head of the individual larva on the right, dorsal side with a needle.

25. The method of claim 24, wherein the piercing comprises about 1 / 3 of the needle for about 2 seconds.

26. The method of claim 24, wherein the needle is between a 22-gauge needle and a 30-gauge needle,27. The method of claim 16, wherein the extracting comprises multiple centrifugations.

28. The method of claim 16, wherein the nucleic acid is selected from genomic DNA or RNA29. The method of claim 16, further comprising rearing the individual larva at room temp after obtaining the biological sample.F& RRefNo.: 55877-0012WO130. The method of claim 29, wherein rearing comprises placing the individual larva in a habitat comprising dry feed; and spraying the dry feed with salt water (3%) daily for about 7 days.

31. The method of claim 16, wherein the amplifying comprises amplifying SEQ ID NO: 9 and / or SEQ ID NO: 10.

32. An article of manufacture, comprising oligonucleotides for amplifying Sequence 1 (SEQ ID NO:9) and / or Sequence 2 (SEQ ID NO: 10).

33. The article of manufacture of claim 32, wherein the oligonucleotides for amplifying Sequence 1 comprise SEQ ID NOs: 1-4 and the oligonucleotides for amplifying Sequence 2 comprise SEQ ID NOs: 5-8.

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