IDRC : Entomology Unit

General Procedure For Submission Of Specimen

Arthropods responsible for the transmission of the agents of human diseases may be submitted to the Medical Entomology Unit of Infectious Disease Research Centre for identification. The Unit also performs tests for the identification of mosquito blood meal, the detection of malaria sporozoites and the isolation of viruses from mosquitoes.

Every specimen must be labelled clearly; it must also be accompanied by a request form giving full information on the following: the habitat or host, the breeding site, the place or locality of collection, the general environment, the date and time of collection, as well as the person collecting the specimen, i.e., the collector.

As identification, especially of different species of mosquitoes, is dependent upon specimens arriving intact at the laboratory, great care must be taken in the handling and preparation of the specimens for despatch. The reader is referred to the relevant sections for instructions on the handling and preparation of appropriate specimens for despatch to the entomology laboratory.

Specimens For Identification And Other Tests: Collection And Preparation

2.1 Bugs - bed, triatomid or other bugs

Kill the bugs in chloroform or ether and wrap them with a piece of tissue paper. Place the packet in a pillbox and send it to the entomology laboratory. Alternatively, the bugs can be sent alive in plastic tubes with sufficient aeration and, preferably, lined at the bottom with a piece of damp filter paper. If entomological pins are available, pin the bugs, through the thorax, onto a cork or styrofoam and send the specimens in a suitable box. Also collect the eggs and nymphs and despatch them to the laboratory together with the bugs.

2.2. Fleas

The most effective way of collecting fleas is to pick them directly from the mammalian or avian host. Catch the host e.g., a rodent, alive and place it in a bag made of cloth or plastic followed by anaesthetizing and killing it with chloroform or ether in a large jar. Next, remove the animal and place it on a white tray or a piece of white paper. Brush the animal hair from tail to head, with a toothbrush or comb. Collect the fleas with a pair of forceps, a fine brush or a dissecting needle that has been dipped into alcohol. Also collect fleas from the bag if there is any. Drop the fleas into 70% alcohol contained in the tube. Fill the tube to the brim with the alcohol, cap it securely, and send to the laboratory.

In the case of cats and dogs, the fleas can be easily obtained from the hairs using a pair of forceps. In infested areas, fleas may be seen hopping onto a piece of white paper or cloth placed on the ground. Pick them up with a pair of forceps and drop them into 70% alcohol contained in a tube. If they are to be sent alive, drop them into a clear test- tube fitted with a cap. Cap the tube and sent it to the laboratory.

2.3. Sandflies, blackflies and other biting midges

Kill the adult flies with chloroform. Using the minutum entomological pins, pin the flies through the thorax onto a cork sheet or Styrofoam. And send specimens in an insect box or other suitable containers.

2.4. Flies

1. Adults:
   Kill the adults with chloroform and place them between layers of tissue paper contained in a pillbox. If entomological pins are available, pin them, through the thorax, onto a cork or styrofoam. Send the specimens in a suitable box to the entomology laboratory.
2. Maggots:
   With the aid of a pair of forceps, collect a representative sample of both small and large larvae and transfer them together with some meat, stool or other materials on which they are found, to a jar with some saw dust or sand in it. Cover the jar with a fine netting of cotton or wire to allow adequate aeration. The adults that emerge will enable confirmation of the preliminary identification based on the maggots. Also kill a sample of the larvae in hot water (60oC) and preserve them in 70% alcohol contained in a universal bottle. Cap the bottle tightly and deliver to the entomology laboratory.

2.5. Lice- body, head or pubic

The adults and nymphs of head or pubic louse can be readily detected by separating the hair with a comb, the eggs (nits) by examining individual hair. Pick the adults with a pair of forceps and drop them into 70% alcohol contained in a tube. Also collect a few strands of hair with eggs attached to them and drop them into the alcohol. Cap the tube securely and send them to the entomology laboratory.

2.6. Cockroaches

Kill the insect with chloroform. Pin the specimen with entomological pin via the thorax onto a cork sheet or styrofoam and dispatch the specimen in a suitable container or box.

2.7. Mites

In adults, the mites, Sarcoptes scabiei, can usually be found in the stratum corneum of the following areas: the clefts between the fingers, the flexor surface of the wrists, the feet, the extensor surface of the elbow, the axilla, the penis and scrotum, and under the breast. In children, the mites can be found anywhere in the skin.

Remove the mite by inserting a needle into the skin under it and lifting it out. For the collection of the eggs, faeces as well as the adult, use a fine sterilized scalpel to remove materials from the area, which gives the greatest itch at the time of collection. Place these materials in 70% alcohol contained in a fine tube or vial. Cap the tube or vial securely and send it to the entomology laboratory.

2.8. Mosquitoes

1. Adult:
   Kill the mosquitoes with a drop of chloroform or ether in a tube. Empty the contents of the tube onto a piece of white paper. With the aid of a pair of fine forceps, pick up the mosquitoes and place them between layers of tissue or lens paper contained in a pillbox - DO NOT USE COTTON IN PLACE OF LENS OR TISSUE PAPER. The number of specimens per layer should not exceed 10. Since identification of mosquito species is often based on the coloured scales on the body, great care should be taken during packing to ensure that the specimens do not come into contact which one another; this is to minimize damage during transport. Also place a few pieces of naphthalene crystals in the box. Close the box securely and send it to entomology laboratory. Live adults can be sent in individual tubes, which must be lined at the bottom with a pledget of damp cotton. Alternatively, they can be sent in a suitable cage covered with a piece of wet lint.
2. Larvae:
   Place the larvae in a porcelain bowl or any other clean container. Kill them with hot water (about 60o C) - DO NOT USE BOILING WATER. Next, pick up the dead larvae with a wide-mouthed pipette and drop them into 70% alcohol contained in a bottle. Fill up the bottle with alcohol, cap it securely and send it to the laboratory.
3. Samples of blood meal:
   This is collected using a piece of filter paper. Divide the filter paper into 8-16 segments (see figure on next page), number the segments and collect each sample of bloodmeal on each segment of the filter paper. Allow the specimens to dry thoroughly before packing the filter paper for despatch. If more than one filter paper is used, the filter paper should be separated from one another with a piece of grease-proof paper during packing; this is to avoid cross-contamination. Place the filter paper in the plastic bag, seal the bag and send it to the entomology laboratory. The following information must be provided with each batch of bloodmeal sample: a) locality/site of collection; b) mosquito species in relation to filter paper samples; c) animal hosts found at the site of collection; and d) date of collection.
4. Specimens for detection of sporozoite:
   Send only specimens of species of Anopheles from areas where active transmission of malaria is believed to be taking place. Place the mosquitoes in a vial, envelope, or any other container. Store them frozen or dry. If the specimens are frozen, be sure to keep them frozen at all the times; this is to reduce bacterial contamination that can interfere with the ELISA test. Mosquitoes from each collection period should be placed in a single container. The following information must be provided with each batch of specimens: a) collection site – kampung, district and state, b) collection method – cattle bait, bare leg collection or other methods, c) date of collection, d) time of collection, e) number of specimen enclosed in each container, and f) species of mosquitoes enclosed in each container.
5. Specimens for virus isolation:
   Place the mosquitoes in a vial or tube. Cap the vial securely, pack it in ice and send it to the laboratory immediately. If delay is unavoidable, store the specimen at 4o C. Note: Different species of mosquitoes should be kept in different containers. The information required for each batch of specimen is similar to that for specimens for detection of sporozoite.

2.9. Venomous and urticarial arthropods

Examples of these arthropods include ants, bees, blister beetles, centipedes, moths, scorpions, spiders, ticks and wasps. These arthropods, whether dead or alive, should be handled with care to avoid damage. Place them in 70% alcohol contained in a screw-capped bottle. Cap the bottle tightly and send to laboratory. If the arthropods are to be sent alive, place them in a suitable container with sufficient aeration to avoid suffocating them; at the same time make sure that they have no means of escape. The container should, preferably, be lined at the bottom with a piece of damp filter paper. Some arthropods, e.g., urticating moths or beetles, can be sent dry. Pack them in fine tissue contained in a box and send to laboratory. If entomological pins are available, they can be pinned, through the thorax, onto a cork or styrofoam and sent in a suitable box to the laboratory.

Figure shows how filter paper should be divided for bloodmeal collection.

* Space for labelling – the filter paper must be labelled with the following particulars:

1. The locality/ site of collection,
2. The date of collection, and
3. The filter paper number if more than one filter paper is used.