Training on ocular identification of Leafminer flies and its natural enemies in farmers' fields

Module 2

Identification Of Leafminers And Their Natural Enemies

Objective: The participants should learn and become thoroughly adopt these simple methods. For insects that are carelessly collected and poorly preserved are practically useless.  

One of the best ways to get acquainted with the different groups of insects is to go out and collect them. Rearing the immature stages to adults and handling and preparing them for preservation and storage will provide many things that cannot be learned from textbooks. 

I. Collecting Insects. Where, when and how to collect insects. Insects have assumed countless range shapes and habits enabling them to adapt with life under almost any circumstances. This vast and amazing group of arthropods can be found practically everywhere and usually in considerable numbers. 

Plants are the best places for collecting insects. Practically all of the plant may harbor insects but the majorities are to be found on the leaves or flowers while others may live in or on the bark, stem, fruits or roots. For leafminers and its natural enemies collect mined leaves and retain them till adult emergence. 

Collecting Equipment. The collecting equipment and other materials depends on the group of insects one is interested in. However, if one intends to make a general collection with no particular group in mind, the equipment described below will suffice for collecting aquatic and terrestrial insects. 

1. Insect nets. Insect nets can be bought from a biological supply house or it can be home made. There are three useful types of insect nets: one for collecting aquatic insects called water dip net; the other for collecting flying terrestrial insects called air net and the third for collecting insects in heavy grass and underbrush called a sweeping net (Figure 7). 

The sweep net is passed rapidly back and forth over the plant canopies/foliage (Figure 8). This method will produce a great quantity and variety of insects. A sweep net can be constructed as follows: The handle should be of hard wood, 1-1/4 inches in diameter and 124 inches long; ring of very stiff and heavy cadmium plated metal or strong wire 12 inches in diameter, and attached to the handle by means of a well-fitted brass ferrule; bag of strong unbleached muslin, 24 inches deep, and reinforced at the top by a double strip of pliable brown leather. The bag should be attached to the ring by means of strong non-corrosive steel wire.

Figure 7 -Assembling an insect net.

Figure 8. An insect net is turned to prevent the escape of captured specimens. 

2. Killing Jars. Insects should be killed in jars containing some poisonous substance of they are intended to be preserved and kept for long. Four sizes of killing bottles are desirable: one should be a six or eight ounce screw-capped jar to be used mainly for insects caught by sweeping, another four to six-ounce jar for lepidopterous insects (butterflies and moths) only; a third should be a heavy glass tube to be used for collecting and killing individual insects from the net; and a fourth, a small tube or vial enough to be carried in the pocket. Adults of leafminers and its natural enemies caught in insect net can be transferred into a plastic bag and kept in the freezer to immobilize and kill them. 

Some of the materials that are used as the toxic agent in a killing jar are potassium, sodium or calcium cyanide, ethyl acetate, carbon tetrachloride and chloroform. Cyanide, however, is the most preferred because it can kill quickly and last a long time. Cyanide crystals and its fumes are extremely poisonous so that great care should be exercised in labeling the jars "Poison" or "Poisonous Fumes". 

Cyanide jars are made as follows: Place about 1/4 - 1/2 inch of potassium or sodium cyanide in the bottom of an eight ounce dry jar and cover with about 1/2 - 3/4 dry fine sawdust. Tamp down gently with a wooden pestle to level the surface and add about 1/8 - 1/4-inch dry plaster of Paris; level it and tamp down tightly. Cut a piece of blotting 

paper of the same diameter as the bottom of the jar. Place the blotting paper on the sawdust and press it down firmly. The blotting paper should be slightly larger than the diameter of the bottle in order to make it firm fit. When the jar has been prepared place a drop of water on the blotting paper with a pipette. The water
starts the reaction of the cyanide; thus producing fumes which kill the insects. A killing jar made of plaster of Paris takes more time to prepare but will last longer. A layer of dry plaster of Paris is placed over the finely granulated or powdered potassium or sodium cyanide instead of sawdust, and then another layer of wet plaster of Paris is poured in. The bottle is left open outdoor until the wet plaster of Paris has thoroughly set and dried which may take a day or two. The bottle is then corked and properly labeled.

3. Vials of preservative. These are needed in collecting soft-bodied and aquatic insects and the immature forms of some insects. The most 

common preservative used is alcohol although other kinds of preservative mixtures are also used (Figure 10). For larvae, pupae, and adults of leafminers preserve them in 70 percent alcohol. In general terms, the more the specimens the better as variations, perhaps peculiar to the region, can be assessed. For molecular (DNA sequencing) analysis preserve in 95-100% ethanol and placed in freezer if they are to be stored for more than a week or so before sending them for identification.

4. Paper envelopes. These are needed for the temporary storage of hard-bodied insects (Figure 11). 

5. Pill boxes or small tin cans. These can be used to store temporarily small hard-bodied insects. The procedure is to place layers of tissue paper, and on each layer are placed insects collected on the same day and in the same locality. A collection data should accompany each layer.

6. Aspirator. If one wishes to catch insects and keep them alive, this device is particularly useful (Figure 13). For collecting adults of leafminers in field on top of leaves, use aspirator before sunrise and after sunset. 

Figure 13. Two types of aspirator assembly

7. Traps. These are devices, which attract and trap insects. Light traps are often used to get data on insect populations; however, insects caught in this manner are oftentimes badly mutilated. Yellow sticky traps are very useful to assess the presence and abundance of leafminer adults and its natural enemies. Please remember they should be used only as detection tools but as a control option.

The collection data. Any collection of insects is scientifically useless without any collection data. The collection data should be entered in a small pocket notebook containing as much information as possible about the specimens to be of scientific value. A more or less desirable data should contain the following information: 

• Locality 
• Date of collection
• Elevation
• Food Plant (if plant-feeding)
• Host (if parasitic)
• Collector's Name 

II. Preserving Insects 

The method of preserving insects varies from group to group. Some insects are mounted on pins and dried or mounted on microscope slides; others must kept in fluid preservatives.

Insects should be mounted on pins the day after they are collected. Flies are best to mount within a few hours after they are captured. If they are kept in a tightly closed jar they remain relaxed for a day. If specimens have been stored for a long time in pillboxes, tin cans or paper envelopes, they must be relaxed before being mounted. A wide-mouth can or jar can be used as a relaxing jar. Place wet sand or cloth at the bottom of the jar or can and a few grains of carbolic acid or phenol to prevent molds. Fit in a screen wire where the paper envelopes, ill box or tin can containing the insects can be propped. The pillbox or tin can should be kept open. Cover the relaxing can or jar tightly, the length of time the insects become relaxed can only be determined through practice though it usually takes a day or two. 

Many of the smaller and more delicate specimens, such as leafminers, should be handled very carefully in order to avoid rubbing off the minute hairs and scales; these are often important in identification, particularly if the specimen is to be identified to species level. The only way to get good specimens of many of these delicate forms is to rear them and to get them into a killing jar without using a net. Leafminer flies are best mounted using cardboard points. They are mounted on sides using nail polish or glue. For larvae, pupae, and adults of leafminers preserve them in 70 percent alcohol. For molecular (DNA sequencing) analysis preserve them in 95-100% ethanol and place them in deep freezer prior to sending them for identification.

Some of the materials needed for mounting insects on pins are the following: 

Pinning boxes. Cigar boxes can be utilized. These are prepared by securely fastening a layer of soft balsa wood, pressed cork or corrugated paper in the bottom of the box. Better pinning boxes can be bought for a small sum from a biological supply house. 

Pins. Special insect pins should always be used in mounting. Insect pins come in different sizes but number three is the most satisfactory size for most insects.

Card board points. These are needed for mounting insects (Figure 14) which are too small for pinning and too large for mounting on slides. They can be made with a special punch using heavy, glazed paper.
 

White shellac, glue or natural nail polish. This is used in attaching insects to the paper points. 

Insect forceps. Insects should always be handled with forceps which may be either a fine-pointed curved forceps or a broad-pointed one (Figure 15a&b). 

Pinning forceps. These are useful in pushing the pins into the cork, but are not absolutely necessary (Figure 15c). 

Labels. Labels should be made from good quality paper. Order and family labels should be outlined with black ink while genus and species labels should be plain. Order labels should be two and one-fourth inches long by one-half inch wide; family labels one and three-fourths by one-half inch and genus and species labels should be one-fourth inch less in length. 

Labeling. The locality should be as small as possible (about 1½ x ¾ cm) and should contain at least the following information: 

• Locality

• Date of collection

• Elevation

• Food Plant (if plant-feeding)

• Host (if parasitic)

• Collector's Name 

This label should be on the pin below the specimen, leaving about the same space between the label and the insect as there is between the insect and the head of the pin. 

For specimens in vials of preservative the same information should be written in India ink on a small bond paper label, which is placed inside the vial. 

The scientific name of the insect written on the label is pinned against the floor of the box or case, which carries the insect. When more than one specimen of the same species is to be mounted, all or the first insect is labeled. 

The order and family labels are placed on the bottom of the case ahead of the insects belonging to that group. 

Care of the Insect Collection. After a collection of pinned insects has been made it should be examined now and then for museum pests. Their presence is readily detected by sawdust like material around the bases of some of the pins. Pouring a tablespoon of carbon bisulphide, benzene, or carbon tetrachloride and keeping the box tightly closed can kill these pests. Since carbon bisulfide is highly inflammable, extreme care should be taken to keep all fires away. Ordinary mothballs will keep the collection free from museum pests but will not kill nor drive them away once they have become established. The best general protecting agent or fumigant is paradichlorobenzene, which is harmless to humans, but actually kills museum pests. A few crystals should be placed in the insect box every three months. 

III. Shipping Insects. 

A. Specimens preserved in fluids. Vials with screw caps or small tubes with rubber or cork covers are used. The container is filled to the brim with preservative and plugged with cotton ball drenched in preservative before the cap or cover is placed to exclude air bubbles. Air bubbles cause fragmentation of small and delicate specimens during transport. If bubbles have been incorporated these can be removed by inserting a hydrodermic needle through the stopper. As the excess fluid comes out through the hydrodermic needle the air bubbles are also drawn out. The stopper seals when the needle is removed and no air bubble is retained inside the vial. If several vials are packed in the same box they should be wrapped with cotton strips or some similar material that no two vials are in contact. The box or container should be of strong material to withstand rough handling in the mail. 

B. Pinned specimens. They should be inserted firmly at the bottom of an insect box. The pins can be prevented from being dislodged during shipment by placing a sheet of cardboard on top of the pinned specimens and the space between this and the lid of the box filled with cotton. 

C. Specimens in pillboxes, tin cans or paper envelopes. These should be packed in such a way that the specimens do not bounce around during shipment. Pillboxes or tin cans should be padded with cotton to immobilize the specimens, and boxes containing paper envelopes should also be filled with cotton. 

D. Slide mounted materials. These should be shipped in wooden or heavy cardboard slide boxes. Strips of soft material should be placed between the slide and the lid of the box, so that the slide does not bounce about. 

Whenever insect materials are shipped by airmail parcel/seamail, an accompanying letter to the addressee should be sent notifying him of the shipment or the letter should be sent ahead of the material. The package sent through the mail should be labeled "DRIED OR PRESERVED INSECTS FOR SCIENTIFIC STUDY" and "NO COMMERCIAL VALUE". Also label the package "FRAGILE".

IV. Rearing Insects. 

The immature stages of many insects are easily reared to the adult stage in the laboratory and some are readily colonized. Almost any kind of a container, preferably a glass or clear plastic jar with a gauze top for ventilation will serve as their rearing container. The proper type of food and substrate (soil, leaf mold, water) are placed in the bottom of the jar. Special care is needed to maintain the proper degree of humidity. 

Activities: 

A. FIELD WORK 

1. Install yellow pan and yellow sticky traps day before field collection. 
2. Collect specimens in the field using the sweep net method and place in killing jars or alcohol vials. 
3. Collect leaves with mines and place in plastic bags/ containers. 

B. LABORATORY WORK 

1. By the use of forceps and brushes, collect specimens from pan and sticky traps. 
2. Sort specimens collected by sweep net method according to group characteristics. 
3. Preserve larval specimens on alcohol properly labeled. 
4. Prepare insect boxes with naphthalene balls 
5. Pin or mount adult specimens on card points. 
6. Place pinned or mounted and vialed specimens properly labeled in insect boxes

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