Insect Taxonomy Lecture 2 - BASIC TOOLS AND GENERAL TECHNIQUES PDF

ENTOM MAJOR 3 INSECT TAXONOMY 2nd Semester, AY 2016-2017 Elorde Jr. S. Crispolon Department of Entomology...

ENTOM MAJOR 3 INSECT TAXONOMY 2nd Semester, AY 2016-2017 Elorde Jr. S. Crispolon Department of Entomology College of Agriculture University of Southern Mindanao From the Lecture of [email protected]/[email protected] Dr. Sheyl A. Yap 09998896432 CPC-UPLB BASIC TOOLS AND GENERAL TECHNIQUES 1. Collecting, Preserving and Storing Insects EQUIPMENTS AND COLLECTING METHODS EQUIPMENTS Forceps a. Fine watchmaker forceps b. Curved metal collecting forceps c. Soft forceps Sample Vials Small containers a. film canister b. plastic box c. metal container Small envelopes/ Paper triangle Absorbent Tissue Notebook A writing equipment Tools for Cutting or Digging Brush Camel’s hair (preferred) Bags Carry-on bags Plastic bags Hand lens Quick identification in the field With lanyard – convenient and prevents its loss while in the field COLLECTING NETS  Aerial net Butterflies Large-bodied flying insects Bag and handle are relatively lightweight Sweep Net Similar to Aerial net Aquatic Net Gathering insect from water Killing Jars Aspirators scene.asu.edu Other Collection Devices Beating Sheets Durable cloth (white) attached to a frame Drag Cloth Durable light-colored cloth attached to a piece of doweling along one edge Sifters Collect insects and mites that live in ground litter, leaf mold, rotting wood, mammal and bird nests, fungi, shore detritus, lichen mosses and other similar materials Separators and Extractors Traps Any device that impedes or stops the progress of an organism With or without baits, lures, or other attractants Depends on its construction, location, time of day or year, temperature, kind of attractant and habits of the organisms Malaise trap Rene Malaise – Swedish entomologist Pitfall and Dish Traps Light Traps Color Traps Bright yellow pan with Manitoba trap – black water-detergent sphere solution Sticky traps Electrical grid traps alibaba.com Baits, lures and other attractants Sugar – attracts ants, moths, butterflies, caddisflies and some flies Fresh feces and other decaying fruits – some flies Oatmeal – crickets, cockroaches and ants Tuna - ants Pheromones – same species of insects Sounds – same species of insects Carbon dioxide - mosquitoes PRESERVATION AND STORAGE OF SPECIMENS Temporary Storage Refrigeration – medium-sized to large specimens – in a tight container – several days – Smaller insects – overnight in a freezer – Use absorbent tissue to keep the specimens dry 36 Temporary Storage Dry Preservation = Small boxes, paper tubes or triangles, or envelopes –NO - Diptera and soft-bodied insects –NO - Tightly closed, impervious containers of metal, glass or plastic –NO – specimen collected at different times or places in the same container –Can be layered in the same container – each layer with data Temporary Storage Papering – Lepidoptera (moths – temporary), Trichoptera, Neuroptera, Odonata and other larger specimens – Butterflies – glassine paper – Do not pack specimens together – No – airtight containers or envelopes Mounting Specimens Pinning Permanent Collection Ideally = fresh specimens Temporarily stored specimens = specially treated before mounting Dry specimens – must be relaxed In liquid preservatives – processed before they will dry Permanent Collection The value of specimens may depend on how well they are preserved. Utility supersedes beauty in research collections. Mounting Specimens May be handled and examined - greatest convenience - least possible damage Enhanced the value of a collection Mounting Specimens Dry specimens – Must first be relaxed – body and appendages will not break, appendages may be rearranged or repositioned – Relaxing chamber – high humidity Beetles –less than 24 hours Small moths and lacewings – 12-24 hrs Large moths and butterflies – 48 hrs or longer - Steam-bathed method Mounting Specimens Degreasing – Grease – internal fatty tissue – Benzene, ethyl ether or carbon tetrachloride – changes color – Vapor degreasing – new technique – trichloromethane – Hexane – very clean and less odor Mounting Specimens Mounting Specimens Liquid-Preserved Specimens – Beetles and some bugs or those with hard exoskeletons – may be directly mounted or pinned without special treatment Mounting Specimens Direct pinning – Refers to the insertion of a standard insect pin directly through the body of an insect  Pinning block Spreading board Orthoptera - back of the thorax to the right of the midline. Large Heteroptera Pin the specimen through the triangular scutellum to the right of the midline. Do not spread the wings. Large Coleoptera Right forewing (wing cover or elytron) near the base. Do not spread the wings of beetles. Large Hymenoptera and Diptera Pin the specimen through the back of the thorax or slightly behind the base of the forewing and to the right of the midline Large Lepidoptera and Odonata Middle of the thorax at its thickest point or just behind the base of the forewings. Spread the wings Mounting Specimens Double mounts – for insects that are too small to be pinned directly on standard pins – minute pin or card points (acid-free) – Adhesive - white glue or carpenter’s glue – soluble in a solvent – Right side of the specimen, with the left side and midventral area clear – Card point - side of the thorax not on the wings Mounting Specimens Slide Preparation Microscope Slide Mounts Mounting meduim Mountants – substances in which a specimen is placed for observations, usually beneath a coverslip for microscopical observation Solid (plastic), semisolid (gelatin), liquid (glycerin), gas (air) Microscope Slide Mounts Mounting medium Water soluble: TEMPORARY Hoyer’s medium Lactic acid Polyvinyl alcohol Glycerin jellies Microscope Slide Mounts Mounting medium Soluble in alcohol: Resins Gum mastic Venice Turpentine Microscope Slide Mounts Mounting medium Soluble in aromatic hydrocarbons: Natural resins (Canada Balsam) Synthetic resins (Permount, Euparol, Histoclad, Piccolytic, Hyrax) Mineral oils (Nuoil) Microscope Slide Mounts Maceration The chemical removal of muscles and other soft tissues while leaving the sclerotized or chitinized parts that are needed for identification 5-10% solution of NaOH or KOH Lactic acid or lactophenol could also be used Careful: chemicals can burn your skin! Microscope Slide Mounts Maceration Time of maceration: dependent on = – Size and physical characteristics of the specimen – Concentration of the chemical agent – Temperature Punctured with a fine needle – agent to penetrate the body Heating could accelerates the action of the macerating agent but CARE must be taken For genitalia – 10-20% KOH OVERNIGHT is recommended Microscope Slide Mounts Washing Distilled water or tap water Add acetic acid – to remove excess caustic substance Dehydration if the mounting medium has a resin base Immerse the specimen in a graded series of EtOH (50% EtOH, 70%EtOH, 80%EtOH, 90% EtOH, 95% EtOH, two washes of absolute EtOH) TIME – varies with size and nature of the specimen Microscope Slide Mounts Staining Define shape – specimens sometimes become colorless Acid fuchsin – aphids, lice and scale insects No stain – Hoyer’s medium Bleaching If specimens are too dark 1 part ammonia: 6 parts hydrogen peroxide sol’n TIME – depend on the amount of bleaching needed Microscope Slide Mounts Temporary mounting May be kept for a year or more Hoyer’s medium – good mounting media – Refractive index is excellent for mites – Water soluble – Tend to crystallize with age (need remounting) Microscope Slide Mounts Permanent mounting Canada Balsam Involves: Maceration, Washing and Dehydration Drawback: It yellows with age LABELING  Collection data should accompany specimens at all times during preparation The label or the information is as important as the specimen No label, no SCIENTIFIC VALUE Paper An appropriate is important – Acid free – Made of cotton fibers – 100% rag content – Should be heavy – Surface of the paper should be smooth Lined ledger paper =100% rag & 36 -pound weight 2-ply Bristol board Herbarium sheets Pens “rapidograph” or technical drawing pens 0.25 mm (no. 000) to 0.30 mm (no.00) Ink India ink Permanent Will not “run” – placed in jars or vials with liquid preservatives Soft lead pencil Lettered and Printed Labels Labels may be lettered carefully by - hand with a fine-pointed pen - printers – Arial, size 4 to 6 Size of Labels - The maximum size of a label should be about 7 x 18 mm - 5 lines or about 13 capital letters - Use multiple labels - to accommodate all of the information that must be presented Label Data - accurate, concise and unambigous Locality – it can be found in any good map – Coordinates of latitude and longitude may be given – Countries: capital letters – U.S.: capital letters = States e.g. PHILIPPINES: North Cotabato, Kabacan, 64km, 14.1667°N, 121.2167°E Label Data Date – Avoid ambiguity – Cite the day, month and year – Day and year = Arabic numerals – Month = Roman numerals – Hyphens or periods can be place in between e.g. 01–XII–2012 01.XII.2012 Label Data Collector – Very useful – Last name = spelled out – Given and middle name/s = initials – Many collectors = Leader’s name followed by et al. e.g. E.S. Crispolon E. S. Crispolon et al. Label Data Other data – Very useful or relevant to the specimens or collector – e.g.: hosts of parasites or host plants = as much as possible do not give vernacular or common names, at least give the genus or family name (Host: Homo sapiens sapiens) – e.g.: details of the habitat (elevation, ecological types and conditions of collection) (1500masl, collected by Malaise trap) Placing the Labels - use pinning block – desired height Direct pinning: – Centered under the specimen – Long axis (length) = long axis of the specimen – Should be aligned transversely at right angles to the axis of the body with the upper margin toward the head Placing the Labels - use pinning block – desired height Double-mounted insects: – pin = center of the right side of the label Placing the Labels Vials or jars: – Large labels that includes all collection data – Do not fold the label – minimize damage and loss of small specimens – Place labels inside the vials or jars – Pencil or ink can be use Placing the Labels Microscope slides: – With pressure-sensitive adhesive – Left side = taxonomic information – Right side = collection information – Mounting medium must be included Placing the Labels Identification labels: Used when specimens are sent to an expert for identification: – All collection data or information must accompany the specimens including field notes if available CARE OF THE COLLECTION (CURATION) Housing the collection A serious collector should consider using standard equipment for housing a collection medium-sized to large specimens – in a tight container – several days - ensures uniformity of container size and style when additions are necessary Housing the collection Liquid Preserved specimens – Vials may stored in racks stoppers not in contact with the liquid expedites rearrangement and examination of the material vials should be inspected frequently Housing the collection Liquid Preserved specimens – Vials may stored in racks vials containing larvae or large insects should have their stoppers replaced by cotton plugs cotton-plugged vials can be placed upside down in a large jar filled with preservative Housing the collection Liquid Preserved specimens – Vials may stored in racks Jars with screw tops or damping lids – IDEAL! Stoppers of neoprene or other synthetic material, GOOD quality cork stoppers – PREFERRED than plastics or rubber! Housing the collection Microscope slides – Slides can be kept in a box Wooden or plastic box Inner side of the box – slotted to hold and separate the slides Slides must be dry Housing the collection Pinned specimens – Can be kept in a box Wooden, plastic or cardboard Bottom – with foam (polyethylene or styro) Protecting specimens from pests and mold Freezing – Safest – 2-5 days at a temperature of -200C to -250C (-4 to - 13F) degrees or colder – Specimens should be dry so that there is no danger of crystallization – Incoming packages should be frozen as received so that any pests hiding in shipping materials are killed Protecting specimens from pests and mold Liquid fumigants – Act faster than solid fumigants – Periodic fumigation of all insect storage boxes is necessary (monthly or quarterly) – Examples: carbon tetrachloride (highly toxic), chloroform (highly toxic), ethyl acetate (less toxic), organic, flammable), ethyl dichloride (less toxic, flammable), methyl bromide (less toxic, organic, ozone depleting chemical), sulfuric flouride (less toxic, inorganic, ozone depleting chemical) Protecting specimens from pests and mold Solid fumigants – Known as repellents – Must be replaced immediately – Example: Naphthalene - may be pinned in a box by attaching the mothball to the head of an ordinary pin If crystals or flakes of naphthalene are used, a small quantity should be placed in a cloth bag or in a pillbox whose top is perforated with tiny holes Protecting specimens from pests and mold Drying – serious problem, especially in moist, warm climates – The specimen should be dried. – Only keeping the collection in a dry place will prevent mold. – Dehumidifier is necessary in a very humid places (e.g. Philippines) HOW TO SAFELY BOX AND SHIP PINNED INSECTS SUGGESTIONS FROM GREGORY A. DAHLEM NORTHERN KENTUCKY UNIVERSITY Pinning Boxes Before adding specimens – check bottom of pinning container! Sometimes the foam bottom is not adequately glued down (small) or not glued in to begin with (large) If this is the case: – Coat bottom with glue – Add foam pinning layer – Add weight – Allow to dry Place support pins around outside  Pin ½ cotton ball in one corner Used to capture a head or leg or other body part if it falls off during shipping Light parts of insects tend to lodge in the soft cotton fibers May be possible to associate part with specimen Cut cardboard inner lid Set the pinning box upside down on a piece of corrugated cardboard Draw shape with pencil Cut out cardboard – should be just slightly smaller than outer edge of box Add specimens Do not pin “densely” If open space remains in box, add some support pins to fill the space Make sure that specimens are securely pinned Add cardboard inner lid Gently set cardboard inner lid on top of specimens Make sure lid is stable and fits well inside pinning box If necessary, add extra support pins in empty areas of pinning box Fold a piece of tape to make a “handle” for easy removal of inner lid Add filler to top of insert You want to make sure that the space between the cardboard insert and box lid are filled Use folded paper towel or piece of bubble wrap to fill space Add lid and label Put pinning box into shipping box half filled with foam “peanuts” – make sure you have at least 2 inch margins on all sides (sides and top/bottom) Fill box to just over rim with foam peanuts When you close and tape down lid, you should have to smash down the peanuts just a bit to get lid to close  Tape, label, and ship Remove all old labels and mark out any old printed addresses or info if you are re-using a cardboard shipping box Tape the box well with good quality shipping tape or strapping tape You may want to wrap box in brown shipping paper, especially if old box has lots of markings on it Use red ink marker to write “Fragile” on several sides of box (or use stickers labeled “fragile”) Include both return address and shipping address on front