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Entomological field
techniques for
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PART I
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Learner’s Guide
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World Health Organization
Geneva
1992
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WHO Library Cataloguing in Publication Data
Entomological field techniques tor malaria control.
Contents pt I Learner s guide — pt 2 Tuto: sgu.de
2. Entomology — education
1 Anopheles
5 leaching materials
methods
ISBN 92 4 154439 2 (pt. U
ISBN 92 4 154440 6 (pt. II)
3 Malaria — prevention and control
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4.Mosquito control —
(NLM Ciassitication QX 18) .
World Health Organization 1992
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publications, in part or tn toto. application should be made to the Oft.ce of Publications. World Health
Organization. Geneva. Switzerland. The World Health Organization welcomes such applications.
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of any opinion whatsoever on the part of the Secretary of the World Health Organization concerning the legal
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boundaries.
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Contents
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30"
Preface
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Introduction
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Learning Units
1. Basic tasks of collecting mosquitos in the field
2. Malaria and its control
3. Role of entomological work in malaria control
4. Recognition of anopheline mosquitos
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5. Hand collection methods and transport of adult mosquitos
6. Hand collection of indoor-resting mosquitos
7. Spray sheet collection of mosquitos
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8. Outdoor collection of mosquitos
9. Direct catches of mosquitos from bait
10. Collecting mosquitos in baited trap nets
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11. Collecting mosquitos from exit traps
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12. Collecting larvae and pupae from breeding sites
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13. Assisting the field supervisor
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Explanation of terms used in the manual
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Preface
This training module on entomological field techniques for malaria control is in
two separately bound parts. The first part is the Learner's Guide, containing
technical information; Part II is the Tutor's Guide, which provides advice for
those responsible for conducting training programmes.
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The module is one of several1 being produced by the World Health Organiza
tion, each concerned with a different aspect of the control of malaria. It can
stand alone as a medium for the training of public health workers engaged in
entomological field work for malaria control, or it can be used as part of a longer
and more comprehensive training programme on malaria. The need for such a
module was identified by the Member States of the World Health Organization,
and it was developed by Mr J.L. Clarke in collaboration with the Programming
and Training Unit of WHO's Malaria Action Programme. The original
illustrations were meticulously prepared by Mr Yap Loy Fong of Kuala Lumpur,
^Malaysia. The text has been revised on the basis of observations and
suggestions made by many people, in particular by Dr McWilson Warren,
former Team Leader of WHO's Interregional Secretariat for the Coordination of
Malaria Training in Kuala Lumpur, Malaysia, and by staff attached to that unit,
notably Mr P. Blizard, Professor C.Y. Chow, Dr Han II Ree, Professor B. Richter,
Mr J. Storey and Dr Suwan Wongsarojana. The text was finally modified by
Dr P.F. Beales, Dr C.W. Hays and Dr D. Muir.
WHO expresses appreciation to all who have assisted in the preparation of the
module, and wishes to acknowledge the collaboration and financial support
provided by the United States Agency for International Development in this
and other activities of the Interregional Secretariat for the Coordination of
Malaria Training.
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‘Already published: Basic nuilaria microscopy (1991).
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Introduction
This Learner's Guide provides teaching material concerned with the entomo
logical techniques involved in the control of malaria. Together with the related
Tutor's Guide, it forms one component — or module — of a series of teaching
materials on malaria. It is designed to be used throughout a formal period of
training and provides information in a simple, easily understandable form.
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The Guide is divided into chapters called Learning Units, which provide you
with all of the essential information you will need in order to apply entomo
logical techniques in the field. The tutor will discuss with you the tasks that you
must be able to perform competently before your training is completed.
Individual Learning Units deal with the theory underlying each task, and the
necessary practical skills involved. Each Unit contains a set of learning
objectives which identify what you should know and be able to do before you
move on to the next Unit.
Your tutor will make use of the learning objectives to develop the content of
the training programme and as a basis for assessing your achievements. You
will find them valuable as a guide for your studies, both as a means of judging
your own progress and as a summary of the key areas in which you can expect
to be tested. For instance, if a learning objective states that you must be able to
“collect indoor-resting mosquitos using a sucking tube" or "kill and preserve
larval specimens", you may be certain that your skill in these tasks will be
assessed.
Careful study of each Learning Unit in advance of related training sessions will
help you to a better understanding of what the tutor is discussing or
demonstrating. You may already have some knowledge or experience of some
of the topics covered, in which case you will have more time to devote to
studying material that is new to you. Many of the terms used in the text are
explained in a section at the end of the Guide.
Because the information provided in the Learner's Guide is so comprehensive,
you will not need to take many additional notes during your training. The
Guide will also be a valuable source of reference after you have completed your
training.
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LEARNING UNIT 1
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Basic tasks of collecting mosquitos
in the field
Learning objective
By the end of this Unit you should be able to:
• define the basic tasks of mosquito collecting
If you are to collect mosquitos in the field it is important that you know and
understand what is expected of you. Your tutor will discuss with you the tasks
involved in field collection, to make you aware of the responsibilities related to
this work. You should feel free to ask questions about anything that is unclear
to you.
Mosquito collection
Mosquitos are usually collected by a team of people working with com
munities, with other health workers, and with laboratory staff involved in the
prevention and control of malaria. The team's activities consist of:
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• collecting adult anopheline mosquitos and sending them to a laboratory for
examination;
• collecting eggs, larvae and pupae of anopheline mosquitos and sending
them to a laboratory for examination;
• keeping accurate, up-to-date records of collections in notebooks, and
ensuring that the same information is also recorded on forms maintained by
the team leader;
• constructing outdoor shelters and trap nets for the collection of mosquitos;
• maintaining collecting equipment in good order so that it is always ready
for field work;
• ensuring that any damage to collecting equipment is promptly reported and
quickly repaired;
• informing householders and village leaders of the reasons for collecting
mosquitos and other specimens;
• answering questions from the community about malaria and referring those
to which the answers are not known to the supervisor or team leader.
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LEARNING UNIT 2
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Malaria and its control
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Learning objectives
By the end of this Unit you should be able to:
• define in simple terms what malaria is
• describe the life cycle of the malaria parasite and the mosquito, and relate this to
the transmission of malaria
• describe what is meant by malaria control
• describe the kind of advice you will provide on vector control, to both individuals
and communities.
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The purpose of this Unit is to inform you about malaria and its control, so as to
help you understand the reasons for the work you will be doing. Read it
carefully and discuss it with the other learners. Your tutor will explain any
points that are unclear to you. Reread the Unit on completion of your training
so that you fully understand the necessity for malaria control and the purposes
of your work.
The work you will be doing can be repetitive, hard and tiring, yet it is
necessary if an acceptable level of malaria control is to be achieved. An
insufficient understanding of the responsibilities of antimalaria work, especially
the team work involved, can have adverse consequences for the health and
welfare of people who live in areas where the disease occurs.
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Malaria — the disease
Malaria is a serious disease and a significant health problem in most tropical
countries. It is caused by a parasite which is found in the blood of people
suffering from the disease. Symptoms of malaria include fever, chills, sweating
and general body pains.
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Malaria parasites are Itransmitted from one person to another by female
mosquitos of the genus; Anopheles, many kinds of which exist in different parts
of the world. Only certain species of anopheline mosquito, known as vectors or
carriers of malaria, can transmit the parasites.
When a mos<;quito bites, it sucks up blood. If malaria parasites are present in the
blood, they are taken into the mosquito where they multiply. After several
days, malaria parasites may be found (in the form of sporozoites) in the salivary
glands of the mosquito. The mosquito also injects saliva when it bites, and can
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Entomological field techniques for malaria control. Learner's Guide
thus transfer parasites to previously uninfected people. Moreover, the parasites
multiply in the red blood cells and destroy them, causing anaemia. This is one of
the reasons why people with malaria may develop a greyish-yellow skin colour.
A person who is sick with malaria can be bitten by many mosquitos every
night, each of which can then carry the disease to several other people.
Moreover, one mosquito can bite many times during its life, and a person who
has been cured of the disease can be infected again if bitten by a mosquito
carrying the parasites. This is why so many people can become sick with
malaria.
A person who becomes infected with malaria can take medicine to kill the
parasites. One of the medicines most commonly given is called chloroquine, but
there are other antimalarial medicines that may be better in certain circum
stances. It is important that people with malaria get the right kind of medicine
from a community health worker or a doctor. If a patient does not take suitable
medicine in the correct amount, at the proper time and for the specified number
of days, the parasites in the blood will survive and multiply and he or she will
become sick again.
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People who have survived many attacks of malaria acquire some immunity to
the disease, even when they take no medicine or an inadequate quantity of
medicine. The number of parasites in the blood decreases and the infection may
produce few, if any, clinical signs and symptoms. There may no longer be any
risk of death from the disease. Consequently, in areas where malaria is highly
prevalent, deaths from the disease occur mainly in children aged betwen 6
months and 5 years, who have had very few attacks. Nevertheless, a mosquito
that bites a person who is resistant to the acute infection can still pass parasites
on to other people.
Certain drugs can also be taken for prevention of malaria, and these arc
particularly important for workers responsible for collecting mosquitos, who
may be bitten frequently. Such workers should take preventive medicine
whenever necessary; trained health workers can prescribe the correct medicine
and dosage.
The malaria parasite
The parasites that cause malaria belong to the genus Plasmodium. Four species
of Plasmodium can live in the blood of humans and cause malaria: Plasmodium
falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. Usu
ally only Plasmodium falciparum can cause death during an acute attack of
malaria. Other kinds of malaria parasite infect animals but are not normally
passed on to humans.
Malaria parasites are very small and can be seen only under a microscope. They
can be observed by examining a drop of blood from an infected person.
Sometimes there are numerous parasites, sometimes few. If there are only a few
parasites, it may be necessary to spend several minutes examining a blood
smear under the microscope before one can be found. Using the microscope, it
is also possible to identify the species of parasite that is present: a trained
worker can determine whether a patient has been infected by Plasmodium
falciparum, or by the other, generally less harmful, species.
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Malaria and its control
Life cycle of the mosquito
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Malaria is transmitted to humans by female mosquitos of the genus Anopheles.
Male mosquitos feed on plant juices and nectar, rather than blood, and
therefore cannot transmit the disease. Moreover, only certain species of
anopheline mosquito are successful vectors of the parasites. Some species prefer
to take their blood meals from animals and thus transmit malaria to humans
very rarely or not at all; some species do not live long enough to allow the
parasites to multiply and develop inside them; and in some species the parasites
seem to be incapable of development.
There are four different stages in the life cycle of the mosquito, of which the
adult is the flying stage. The adult female anopheline takes a blood meal
approximately every two or three days. This is necessary for the development
of a batch of eggs, which are normally laid before a further blood meal is taken.
The time required for digestion of the blood meal and development of the eggs
varies with the temperature and humidity of the air.
Eggs are laid on water in batches of about 100-150. The sites for egg-laying
vary from small amounts of residual water in such places as discarded food tins,
coconut husks and hoof-prints, to larger areas of water in streams, canals, rivers,
ponds and lakes. Each species of mosquito prefers a particular kind of water
surface on which to lay its eggs.
Female mosquitos continue to lay eggs throughout their lives. Most lay
between one and three batches, although in a few instances as many as 13
batches have been reported. Under the best conditions the average life of
female anopheline mosquitos is about two to three weeks.
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After two to three days, the eggs hatch and the mosquito larvae emerge. The
larvae generally live just below the surface of the water (since they need to
breathe air) and take food from the water. If disturbed, most larvae swim
quickly towards the bottom of the water, returning to the surface soon
afterwards in order to breathe; certain species, however, move rapidly sideways
just below the surface.
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There are four larval stages or instars. The time required for the different stages
to develop depends on various factors, including water temperature: the
development period is shorter in warm water than in cool water.
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The small larva emerging from the egg is called the first instar. After one to
two days it sheds its skin and becomes the second instar; at further intervals of
about two days the third and fourth instars appear. At normal tropical water
temperatures, the larval stage thus lasts about 8 to 10 days. Finally, the fourth
instar develops into a pupa.
It is during the pupal stage, which lasts for two or three days at tropical
temperatures, that the major transformation takes place, and the aquatic
organism changes into an adult mosquito capable of flight. The pupa, shaped
like a comma, stays on the surface of the water and swims when disturbed but
does not feed. Its skin then splits and the adult insect emerges and eventually
flies away. Mating soon follows; the female, which mates only once, normally
takes its first blood meal after mating and then develops its first batch of
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Entomological held techniques lor malaria control: Learner's Guide
Most anopheline mosquitos bite during the night, some at sunset, others
around midnight. Some enter houses to bite, while others usually bite outdoors.
After biting, mosquitos usually rest for a short period. Indoor-biting mosquitos
frequently rest on a wall, under furniture or on hanging clothes. Mosquitos that
bite outdoors may rest on plants, in dark holes in the ground, or in other cool
dark places.
Mosquitos may bite both people and animals. Host preferences differ between
the species: some mosquitos prefer to take blood from humans, others take only
animal blood. Clearly, those that prefer human blood are the more dangerous
as they are more likely to transmit diseases, notably malaria. The incidence of
malaria transmission may also vary with seasonal factors such as temperature,
rainfall and humidity, all of which have a significant influence on the population
of anopheline mosquitos.
Malaria surveys and surveillance
Information on the epidemiology of malaria is essential if the disease is to be
controlled. Some may be acquired through malaria surveys conducted in the
field with laboratory support, following a review of data from previous surveys
and other relevant research. However, the information provided by a survey of
this nature relates only to the situation at one particular time in a specific
country or region. Continuing surveillance., on the other hand, provides a
steady flow of information.
The information collected during surveys should provide answers to at least the
following types of question:
• Is there malaria transmission in the country or region? If so, in which specific
areas does it occur? What are the geographical limits (including limits of
altitude) of the disease? Is malaria absent from large cities?
• What is the prevalence of malaria in the area and how severe is the disease?
Is there falciparum malaria (the most dangerous form of the disease)?
• Is there evidence of P. falciparum being resistant to chloroquine and/or other
antimalarial drugs?
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For entomological purposes, a preliminary malaria survey should determine:
• which anopheline species are present;
• which of them are vectors of malaria;
• the biology and behaviour of adult vector mosquitos, including their resting
habits indoors and outdoors, feeding habits, seasonal changes in the
numbers biting humans, duration of adult life, and areas in which they are
found;
• the breeding habits of the mosquitos;
• which vectors are susceptible or resistant to insecticides.
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Vector control programmes for limiting the transmission of malaria in particular
areas should be planned on the basis of this type of information.
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A full malaria survey is needed only if data are unavailable, inadequate or
unreliable. It may take several months or years to complete a full survey and
this can be quite costly. Any such survey must be planned and directed by an
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Malaria and its control
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experienced malariologist, who can bring together information from para
sitological and entomological services, together with data concerning the social
habits, occupations and movements of the people, the types of houses they
live in, availability of transport, climatic conditions, and the extent and
distribution of health services. A partial or “updating" survey or a continuing
(e.g. monthly) system of surveillance may be restricted to some of the items of
a full survey, with a view to supplying missing data, assessing the results of any
malaria control activities since a previous survey, or evaluating and/or
replanning a programme.
Thus a limited survey, by means of malaria microscopy, may be needed to:
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• determine changes in the number of malaria infections and in the propor
tions of the various kinds of causative parasite, with particular reference to
P. falciparum;
• determine any variation in the effects of drugs used against P. falciparum
infections.
For entomological purposes a limited survey may be necessary to:
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• determine whether, following the use of vector control measures, there has
been a reduction in the number of vectors biting people;
• determine whether there has been a reduction in the breeding density of
vectors in the area;
• test the susceptibility of vectors to insecticides.
Once a control programme is in operation, regular observations are made to
determine whether it is achieving its objectives. Mosquito collections are
usually made at the same locations every two weeks, or at least monthly, so that
any changes in vector numbers or behaviour can be observed and recorded.
Random collections can also be made at other sites to confirm the results from
regularly surveyed locations.
In some control programmes, spot checks; or special parasitological and
entomological studies are conducted. If there is a sudden outbreak of malaria in
a village, such studies are often undertaken to determine why it occurred and
what emergency measures against the vector and/or the parasite are needed to
control the outbreak and prevent a recurrence.
Malaria control
In areas where many people have malaria it is important to lessen the burden
on families and the community by treating the sick, preventing mosquitos from
biting people, and possibly also killing mosquitos. In all areas, a combination of
the first two approaches is needed to reduce the amount of suffering and
disease. The third measure should be added whenever feasible in order to
control the intensity and spread of malaria.
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The treatment of malaria involves the use of specific drugs. Dosages for infants,
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children and adults vary according to body weight, but drugs are always given
for a specified number of days. Giving too much of a drug can have unpleasant
side-effects, but if too little is given there may be no effect on the malaria
parasite. It is especially important that the proper treatment be giiven to people
infected with P. falciparum; without it, they may die. Drugs ma;y be given by
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various people, such as village health workers, dispensary health workers,
hospital doctors, trained members of health staffs, trained volunteers, or
members of malaria teams.
Administration of drugs to prevent people from becoming infected with
malaria is called malaria prophylaxis. Prophylaxis is normally restricted to
specific groups of people and given for limited periods of time. If prophylaxis is
continued for long periods in a population, P. falciparum in the area may
become resistant to the drug or drugs being used, which could deprive the
health services of their only means of curing severe infections and result in
deaths. Furthermore, drug prophylaxis is expensive and full coverage is difficult
to sustain for extended periods.
In addition to the treatment of malaria, much can be done to reduce the number
of anopheline mosquitos by eliminating breeding sites and killing larvae, pupae
and adult insects.
Breeding sites can be eliminated by:
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drainage or filling of areas where water collects;
modifying the preferred habitats of particular vector species, for example by
clearing streams so that the water flows faster and areas of vegetation are
eliminated.
Larvae can be killed at breeding sites by spreading oil or other substances on
the water or by introducing fish that eat the larvae.
Spraying houses internally with insecticide is the principal means of killing
adult mosquitos that bite indoors. DDT is generally used for this purpose
provided that the anopheline mosquitos that carry malaria in the local area
show no significant resistance to the insecticide. If, having bitten someone who
has malaria, a mosquito then rests on a wall sprayed with insecticide, it is killed
and thus cannot transmit the disease to other people. This is why, in many
countries, houses are sprayed every six months with DDT or every three
months with a less persistent insecticide.
After several years of spraying with DDT or other insecticides, mosquitos and
other insects may develop a capacity to survive contact with one or more of
them. This insecticide resistance may develop slowly or quickly and range in
degree from slight to total. It is important to know when resistance develops in
one or more vector species because it may result in the loss of effectiveness of
an insecticide in the control of malaria transmission. It is possible to measure
both developing and established resistance, and this can help in deciding
whether an interruption of spraying or a change of insecticide is required.
Various types of personal protection can be used to prevent biting by
mosquitos that carry malaria. These include:
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the use of untreated or insecticide-impregnated bednets at night;
the use of mosquito repellents or mosquito coils;
the fixing of screens on windows to prevent mosquitos from entering
houses;
the use of insecticide-impregnated curtains on windows or doorways and/or
in gaps between the roofs and walls.
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LEARNING UNIT 3
Role of entomological work in malaria
control
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Learning objectives
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By the end of this Unit you should:
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• understand entomological surveys and monitoring and the purposes of these
activities
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• know how to prepare and maintain accurate records of all such activities.
Entomological services have several important roles to play in malaria control,
including the following:
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• identification of the vectors responsible for transmission of the disease;
• provision of basic information on vector species for planning purposes;
• monitoring the impact of control measures (for example, by determining
changes in vector population density, rates of infection, susceptibility of
vectors to insecticides, and residual effects of insecticides on treated surfaces);
• contributing to the investigation of problem areas where control measures
prove unsuccessful;
• assisting in training.
When a control programme uses anti-vector measures, the first indicators of its
effects can be monitored by carefully selected entomological observations.
Reductions in vector density, parous rates and other parameters point to
successful control. Further evidence of the success of malaria control may be
obtained by malariometric spleen and parasite surveys and routine epi
demiological observations.
It is important for all results to be accurately recorded and for at least two
copies to be kept separately in case one is lost. Additional copies are required
for transmitting data to other appropriate sectors of the health system.
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Types of mosquito survey
Mosquitos brought to the laboratory are obtained in different kinds of field
survey, each of which has an important role in the planning, operation or
evaluation of anti-vector programmes.
Preliminary surveys
Preliminary surveys are the original, basic, short-term surveys used to gather
baseline data for planning anti-vector measures. They provide information on
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Entomological held techniques lot malaria control: Learner's Guide
the following matters for each locality:
the anopheline species present: specimens are obtained in the field and
identified in the laboratory;
• determination and/or confirmation of vectors: specimens obtained in the
field are examined in the laboratory for infection with parasites;
• the ecology of adult vectors, with reference to such factors as their resting
habits, feeding habits (as determined by human-vector contact studies, and
identification of blood meal sources), seasonal prevalence, and longevity;
• larval habitats, with reference to the types of water accumulation used as
vector breeding sites, in relation to their size, duration, movement, chemical
and physical nature, associated animal and plant life, exposure to sunlight,
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etc;
susceptibility tests, intended to measure the sensitivity of vector species to
available insecticides and to facilitate the selection of the most cost-effective
insecticide.
Regular or trend observations
These are routine or long-term observations, and they are sometimes called
operational surveys or monitoring. Observations' are carried out regularly, e.g.
weekly, fortnightly, monthly or half-yearly, for the purpose of evaluating the
impact of control measures. They should provide information on the following
matters:
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reductions or increases in vector density;
changes in vector biology and behaviour, with reference to survival, feeding
habits, resting places, degree of man-vector contact, etc;
numbers and proportions of vector species with sporozoites in their salivary
glands;
proportions of vectors caught in sprayed houses which survive for 24 hours;
susceptibility of vectors to insecticides.
Spot checks
Spot checks, which may include selected items from the regular observations
listed above, are carried out in localities chosen at random. Since the fixed
stations often used to monitor mosquito populations may not be representative
of all areas, spot checks may be conducted randomly in selected areas to
supplement routine observations or obtain a clearer indication of the effects of
control measures.
Foci investigations
Foci investigations, which are similar to those outlined above, are undertaken in
areas of new or persistent malaria transmission to determine why there is
transmission or why the disease is not responding to the measures being
applied, and to identify the best approaches to control.
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LEARNING UNIT 4
Recognition of anopheline mosquitos
Learning objectives
By the end of this Unit you should be able to:
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• distinguish mosquitos from other insects on the basis of their external character
istics
• distinguish between male and female mosquitos
• distinguish female anopheline mosquitos from female culicme mosquitos
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© distinguish between the eggs of mosquitos and those of other insects
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distinguish between anopheline and culicine eggs
• distinguish mosquito larvae from the larvae of other insects
• distinguish between anopheline and culicine larvae
• distinguish mosquito pupae from the pupae of other insects
• distinguish between anopheline and culicine pupae.
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The collection of adult mosquitos and of their eggs, larvae and pupae forms a
major part of entomological field work In the places where you will typically
undertake collection, you are likely to find many other types of insect as well as
mosquitos. You must therefore be able to distinguish between mosquitos and
other insects. More importantly, since malaria is transmitted to humans only by
mosquitos of the genus Anopheles, it is essential for you to be able to
distinguish between anopheline and culicine mosquitos. You must also be able
to identify anopheline mosquitos at all stages of their life cycle — eggs, larvae
and pupae, as well as adults.
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Recognition of adult mosquitos
The adult is the easiest stage to identify in the life cycle of the mosquito. Fig. 1
shows the main parts of the adult mosquito, and you should learn the names of
these. The body, as in all insects, is divided into head, thorax and abdomen.
Three characteristics can be used to distinguish adult mosquitos from other
insects:
• there is only one pair of wings
• there is a long proboscis (the tubular mouthparts)
• the body is covered with scales.
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Entomological field techniques lor malaria control: Learner's Guide
y-------- Proboscis
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Head —
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Wing
Thorax —
Abdomen —
Legs
Fig 1
-
A mosquito
Fig. 2 illustrates a number of common insects. On the drawing of the adult
mosquito, note particularly the long; mouthparts (proboscis), the single pair of
wings, and the three pairs of corn}paratively long legs.
Distinguishing female mosquitos from males
Because only the female mosquitos take blood meals and transmit malaria, it is
females that you will be required to collect in the field. It is therefore important
to be able to distinguish female mosquitos from males.
The male has very long hairs on the antennae, which consequently have a
bushy appearance, rather like a moustache. On the antennae of the female the
hairs are few in number and short. You should compare the male and female
mosquitos illustrated in Fig. 3.
Distinguishing female anopheline mosquitos from female culicines
With live mosquitos, you can distinguish between anopheline and culicine
mosquitos by observing their resting postures. Anophelines rest at an angle
between 50° and 90' to the surface whereas culicines rest more or less parallel
to the surface. These resting postures are shown in Fig. 4.
20
Recognition of anophehne mosquitos
-i ’
A. No wings
a
-1
termite
silverfish
0
r
AJ
head louse
flea
bedbug
B Two pairs of wings
i
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moth
cockroach
2 3
C. One pair of wings
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i 3
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3
ffl
3
’
3
4
horsefly
housefly
midge
Note: Line scales indicate actual lengths of insects.
" - sj
Fig. 2
21
Common insects
mosquito
Entomological field techniques for malaria control. Learner's Guide
antenna
not bushy
f palp as long
as proboscis
proboscis
J
u
palps
palp very
short
Female anopheline
Female culicine
tip club-shaped
tip not club-shaped
I
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HF
lF
antenna
bushy
Male culicine
Fig 3
JVvt -
—
proboscis
\w
Male anopheline
Heads of male and female anopheline and culicine mosquitos
Anopheline mosquitos can also be distinguished from culicines by the length
and shape of the palps. The differences, which can be seen in Fig. 3, are as
follows:
Female: anopheline — palps as long as proboscis
culicine — palps very much shorter than proboscis.
Male:
anopheline — palps as long as proboscis, club-shaped at tip
culicine — palps longer than proboscis, with tapered tips.
Distinguishing mosquito eggs from those of other insects
The eggs of anopheline mosquitos and of some culicines float on the surface of
water; those of other insects do not float but are found attached to plants or
under stones or other objects. The eggs of some Aedes mosquitos may be found
in places that are liable to dry up; they can survive those conditions for some
time.
22
it
Recognition of anophelirie mosquitos
Distinguishing between anopheline and culicine eggs
To distinguish between anopheline and culicine eggs you should observe
whether they clump together in a "raft” (Cw/fx) or float separately (Aedes), and
whether they have “floats" (Anopheles). It is important that you learn these
differences, which are illustrated in Fig. 4.
1
3 >3
Culicine
Anopheline
Culex
Aedes
float
/
Egg
water
surface
Larva
•iv
£
Culex
=!*
breathing
trumpet
Pupa
water
surface
3
3
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At
Adult
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9
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3
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9
9
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Fig. 4
23
Life cycles of mosquitos
Entomological held techmoues for maiana control. Learner's G<."Of
Distinguishing mosquito larvae from the larvae of other insects
Mosquito larvae differ from the larvae of other insects in the following
respects:
they remain just beneath the surface of the water (because they need to
•
breathe air through a special breathing tube);
•
the head is narrower than the thorax.
Distinguishing between anopheiine and culicine larvae
In the culicine larva the breathing tube is expanded and extended to form a
“siphon". In the anopheiine larva there is no siphon.
In Fig. 4 you will see that the siphon allows the culicine larva to hang down
some distance from the water surface, whereas the anopheiine larva rests
parallel to and immediately below the surface.
You can therefore distinguish anopheiine from culicine larvae because:
the anopheiine larva has no siphon and rests parallel to the water surface;
the culicine larva has a long siphon and hangs down from the water surface.
•
•
Look again at Fig. 4 and study these differences carefully.
Distinguishing mosquito pupae from pupae of other insects
You must be able to distinguish mosquito pupae from those of other insects.
Mosquito pupae, shown in Fig. 4, have the following characteristics:
•
•
•
they are comma-shaped
they hang just below the water surface
they swim actively when disturbed.
Distinguishing between anopheiine and culicine pupae
It is relatively difficult to distinguish anopheiine from culicine pupae in the field.
You should therefore collect all mosquito pupae and take them to the
laboratory for identification. As can be seen in Fig. 4, the breathing trumpet of
the anopheiine pupa is short and has a wide opening, whereas that of the
culicine pupa is long and slender with a narrow opening.
24
%
r*'
LEARNING UNIT 5
*
Hand collection methods and
transport of adult mosquitos
Learning objectives
By the end of this Unit you should be able to:
. identify and assemble the parts of a sucking tube
2^
. prepare paper cup containers for collections
. collect and transfer mosquitos, using a sucking tube
. label paper cups and make records of collections, accurately and complete y
%
. collect mosquitos and transfer them to a paper cup, using a test-tube
4^
. keep mosquitos alive in paper cups under field conditions
- q
• transport live mosquitos from field to laboratory
<1 4
• construct a killing tube
• demonstrate the proper care and cleaning of equipment.
= »«
One of the principal activities in entomological work is the^ fidd collection of
U
t
p J ntoir h-ansnort to the laboratory for identification anu prolo field eolleeHng techniques this
»U teach
3
£ 3
■3
to the laboratory.
At the end of this Unit you will make a
practice in the collection of mosquitos.
killing tube in preparation for field
3
r 3
3
3
Xa^eX^Tt XXi ~ heeonte pcohcent
f
3
the collecting methods that you will use.
f
3
be dissected: dead mosquitos become
3
25
1
al,
Entomological field techniques for malaria control: Learner s Guide
for dissection. The laboratory will:
• dissect out the salivary glands and gut and examine them for evidence of
infection with malaria parasites;
• dissect out the ovary and examine it in order to determine the parous rate;
• carry out susceptibility tests;
• determine the 24-hour mortality rate for mosquitos collected from sprayed
houses.
In this first Unit of the practical part of your training, the main emphasis is
therefore on collecting mosquitos by hand and keeping them alive until they
reach the laboratory. The techniques that you will be taught involve the use of
three pieces of equipment:
• sucking tube
• test-tube
• killing tube.
In the interest of brevity we shall refer, for the most part, simply to "mosquitos",
it being understood that most of your collecting will in fact concern
Anopheles and that the most important information required will relate to the
species that carry' malaria from person to person.
Essential equipment
The following equipment is needed for your activities: sucking tube; torch
(with spare bulb and batteries); unwaxed paper cups; mosquito netting; cotton
wool; mosquito cages; test-tubes; rubber stoppers for test-tubes; rubber bands;
insulated picnic box; chloroform; trays; beakers; towels; pencil; notebook.
Test-tubes should normally be 150 mm long and 16 mm in diameter, but
smaller ones (100 mm x 10 mm or 60 mm x 10 mm) may also be used. Small
tubes are useful for collecting single specimens or when specimens are to be
kept for some time before being examined and processed.
Identifying and assembling the parts of a sucking tube
The type of sucking tube illustrated in Fig. 5 consists of four parts:
•
•
•
•
a length of glass or plastic tube
a disc of wire mesh incorporated into the tube during manufacture
rubber or plastic tubing, 80-100 cm long
a plastic mouthpiece.
A sucking tube can be made locally using glass or transparent plastic tube. Wire
mesh between the collecting tube and the rubber tubing is necessary to keep
the mosquitos in the collecting tube and prevent their being sucked into the
mouth and swallowed. (Precaution: plastic tubing should not be exposed to
chloroform, which makes it lose its transparency.)
Sucking tubes and test-tubes should be kept clean. If they become dirty, clean
them with wet cotton wool and ensure that they are perfectly dry before use.
They should be carried in a way that will prevent breakage.
26
Hand collection methods and transport of adult mosquitos
wire mesh/gauze to prevent
mosquitos being swallowed
glass or plastic tube
■
;
mouthpiece
^^223^
:! *
rubber or
plastic tube
(80-100 cm)
Fig 5 Sucking tube or aspirator
Preparation of paper cups as containers
^•3
-i
1
Cups made of unwaxed paper are very suitable containers for holding and
transporting live mosquitos with minimum mortality. When these are bought
in bulk they are very cheap and can be frequently replaced. Never reuse cups
that have held mosquitos collected from sprayed houses, as residues of
insecticide may kill mosquitos that you want to keep alive.
A paper cup container is made easily as shown in Fig. 6. It consists of:
• an unwaxed paper Cup (capacity 250-300 ml)
• a square of mosquito netting with a hole for insertion of the sucking tube
• a rubber band
• a piece of cotton wool as a stopper.
<3
-a
• ^3
4
•a
-S
If suitable paper cups are not available, small mosquito cages can be used or
containers can be made from other locally available materials.
It is important to ensure that the hole in the netting over the cup is only large
enough for the sucking tube to be inserted. If this hole is too large the
mosquitos can easily escape or become damaged in the attempt.
hole
cotton
wool
--------- rubber band
Sj&x------ netting
SA
5W8
ill
*
Fig 6 Paper cup prepared as a mosquito container
SA
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27
Entomological field techniques for malaria control: Learner's Guide
Use of the sucking tube
When you wish to collect a mosquito and transfer it to a paper cup by means of
a sucking tube, proceed as follows:
• With the mouthpiece in your mouth, hold the sucking tube with its opening
1—2 cm away from the mosquito.
• Move the end of the sucking tube closer to the mosquito and, at the same
time, suck gently but quickly so as to draw the mosquito into the tube.
• Place your finger over the tube to prevent the mosquito from escaping.
• Place the end of the tube, with your finger still in position, near the hole in
the mesh covering the paper cup. Remove your finger and quickly put the
tube into the hole.
• Blow gently into the mouthpiece so as to transfer the mosquito to the paper
cup; at the same time, tap the tube with your index finger to disturb resting
mosquitos.
• Use the same technique to transfer mosquitos between different containers.
Be careful not to suck or blow too hard, as mosquitos are fragile and can easily
lose legs or be otherwise damaged. Do not collect more than five mosquitos in
the sucking tube before transferring them to the paper cup; if they collide with
one another or with the tube they are likely to be damaged. Identification of
species is important and may be hampered if specimens are damaged.
Labelling paper cups and recording collections
Accurate and complete labelling of paper cups is extremely important. During a
field trip, mosquitos are collected at many sites. In the laboratory it is
impossible to know the sources of collections if cups are unmarked or not
labelled completely and correctly. Labelling should be done in soft pencil.
A record must also be kept in your notebook or on a field form of all collections
made during a field trip. Particular attention should be given to keeping a
record of all non-productive
that yields
productive collecting efforts as well as of' collecting
"
mosquitos.
The data you should record will vary with the purpose of collecting, but the
minimum essential information is the following:
i
•
•
•
•
•
location
date and time of collection
type of structure (house, stable, outdoor shelter, etc.)
whether the structure has been sprayed and, if so, when this was last done
your name.
Collecting mosquitos using a test-tube
A test-tube can be
I used to collect mosquitos by the following method, which is
illustrated in Fig. 7.
•
•
•
•
28
r
Hold the mouth of the tube directly over the mosquito.
When the mosquito is disturbed it will fly into the tube.
Close the mouth of the tube with your index finger or thumb.
Remove your finger and push a plug of cotton wool into the tube.
4■
Hand collection methods and transport of adult mosquitos
u
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Wrong way
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€ C9
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Right way to take
test-tube towards
mosquito
£ J
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11
3
£
£ ■3
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Fig 7 Using a test-tube for mosquito collecting
Push the plug down until the mosquito is trapped in the bottom 2 cm of the
tube.
• Collect a second mosquito as described above and insert a second plug to
trap it in the next 2 cm of the tube. In this way, several mosquitos may be
collected in one test-tube.
• Record on a slip of paper all the data described above. Put the paper in the
•
*•3
sea
tube.
29
Entomological held techniques for malaria control: Learners Guide
Keeping mosquitos alive in the field
Field collections must be sent to the laboratory as soon as possible. If
mosquitos are to be kept for some time in the field, precautions must be taken
to keep them in good condition. Remember that dead mosquitos dry out and
become brittle, and cannot then be dissected.
In order to keep mosquitos alive in paper cups it is necessary to take the
following steps:
• Soak pieces of cotton wool in 5-8% sugar solution.
• Squeeze out any excess sugar solution and place the cotton wool over the
tops of the cups.
• Place cups holding mosquitos upright in a deep tray, a cardboard box or,
preferably, an insulated picnic box.
• Cover the cups with a damp towel. Keep the towel damp until the
mosquitos reach the laboratory.
If mosquitos have to be kept in the field for some time and an ant-proof
container such as an insulated picnic box is not available, they can be
maintained in the cups as shown in Fig. 8.
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paper cups
with mosquitos
damp towel
beakers or
glasses
tray filled
with water
Fig 8 Keeping mosquitos alive in the field
Make sure that you keep your mosquitos in places that are free from insecticide
contamination.
Transport of live mosquitos
Adult mosquitos collected in the field have to be transported to the laboratory
for examination. It is your responsibility to make sure that they arrive alive and
undamaged.
Mosquitos can be transported safely over long distances in unwaxed paper
cups provided you pack them as follows:
•
•
•
•
•
30
Cover the cups with damp cotton wool.
Place the cups upright in a box, preferably an insulated picnic box.
Pack newspaper or other material between the cups to minimize movement.
Place a damp towel over the cups.
Close the box as tightly as possible to prevent loss of moisture.
Hand collection methods and transport of adult mosquitos
%
During the period of transportation, it is essential to make sure that the
mosquitos are not left in a vehicle with the doors and windows closed, or in
direct sunlight.
M3
2- £
1 ‘3
^1;
- L.
Constructing a killing tube
A killing tube, as illustrated
illustrated in
in Fig.
Fig. 9,
9, can be used to collect mosquitos for
taxonomic studies or for other purposes not requiring live specimens. It is made
from an ordinary test-tube in the following manner:
•
•
Place pieces of cut rubber band at the bottom of a test-tube.
Cover the rubber bands with a thin layer of loosely packed cotton wool and
•
•
add a circle of filter paper.
Pour chloroform into the tube to soak the rubber.
Close the tube tightly with a rubber stopper to prevent evaporation of the
•
chloroform; pour off excess chloroform the following morning.
When the tube ceases to be effective, soak the rubber with chloroform again.
rubber stopper
=■<!
7/////A
•' I
test tube
^<3
I
^•3
^•3
----------- filter paper
/
.---------- - cotton wool
^•«i
rubber bands
soaked with chloroform
E: 3
Fig 9 Killing tube
& SB
Cleaning and care of equipment
It is essential to keep all equipment:
9
•
•
•
31
free from dirt
free from deposits made by mosquitos
free from insecticide contamination.
Entomological field techniques (or malaria control: Learner's Guide
Your sucking tube must be cleaned frequently and thoroughly. Its component
parts should be separated and washed with water and detergent. The inside of
the glass can be cleaned with damp cotton wool on the end of a thin rod. Each
part must be completely dry inside before the sucking tube is reassembled.
Paper cups are inexpensive and should be regularly replaced. They must always
be thrown away after use in insecticide-treated areas. The mosquito netting
covers for the cups should be washed and dried.
The containers used for transporting the paper cups should be regularly
washed with detergent. If cardboard containers are iused they should be
regularly replaced.
32
1
LEARNING UNIT 6
-i#
*
Hand collection of indoor-resting
mosquitos
Learning objectives
J
By the end of this unit you should be able to:
• explain to householders your reasons for asking to enter their property
• use a sucking tube to collect indoor-resting mosquitos
• observe the normal resting places of mosquitos in houses and associated
buildings
• accurately and completely label paper cups and prepare records of collections
St]
• prepare collections and transport them to the laboratory.
During this Unit of your training programme, you will go out to the field and
meet the local community for the first time. The methods of approaching
people and explaining your intended work to them will be described to you in
advance.
i ''3
You will have the opportunity to practise the collecting methods you have
learned in the laboratory and to observe the normal daytime resting places of
mosquitos. You will also prepare mosquitos and transport them to the
laboratory.
-5
3
*
1 *3
Many of the anopheline species that are malaria vectors rest indoors during
the day and after biting. Hand collecting provides the following important
information about these mosquitos:
•
•
•
•
their usual resting places
their distribution on different types of surface
their indoor resting density
seasonal changes in their density.
Hand collecting can also provide live specimens for:
•
•
•
•
•
susceptibility and bioassay tests
the production of eggs
observations on mortality among insects from insecticide-treated houses
taxonomic studies
laboratory studies on rates of infection and survival.
Information on density obtained by hand collecting is less reliable than that
derived from pyrethrin spray sheet collecting, which will be described in
Learning Unit 7.
<•3
!
33
Entomological field techniques for malaria control: Learner s Guide
Essential equipment
In order to carry out hand collection in the field you will require the following
equipment: a sucking tube; a torch (with spare bulb and batteries); paper cups
with net covers; cotton wool; a pencil; and a notebook.
Your personal equipment should always be maintained in good or^r
kept
in a baPg ready for the field. You must pack glass items carefully so that they do
not get broken while you are travelling.
You will also need the following additional equipment for .the transport of
specimens from the field: insulated picnic box (or other suitable con amerk
sugar solution (5-8%); paper cups with net covers; cotton wool; towels; and
newspapers to serve as packing material.
Communicating with village leaders and householders
Most of your mosquito collecting will be done in and around houses. It is
therefore essential that you establish and maintain good relations with
householders, village leaders and other members of the
PTe~
liminary discussions between village leaders and supervisors will be held before
collecting starts.
You must ask permission to enter each property in which you desire to collect
mosquitos The tact needed and the method of approaching householders wi
be discussed with you by your course tutor. The following are some basic
requirements.
Do:
• observe local customs (e.g. removal of shoes before entering houses)
• respect places of worship and shrine rooms
• respect the privacy of householders
. give any assistance you can to the villagers (e.g. provide advice on mosquito
control and personal protection measures).
Do not:
• make excessive noise or create disturbance
• enter a property without permission
• enter a bedroom unless the occupants are awake and dressed
• ask for personal favours
• ask for food (you may accept food only when it is given voluntarily)
• disturb furniture
• open cupboards
• break anything.
Use of sucking tube to collect indoor-resting mosquitos
You have been instructed in the use of the sucking tube and practised with it in
the laboratory. The skills you have learnt can now be applied in the held.
34
I
Hand collection of indoor-resting mosquitos
J
1 'i
In any village about 10 houses should normally be examined in order to provide
a representative sample. The selection of houses for routine collecting is a
1
1 :1
complex statistical procedure that should be carried out by a competent person.
In the case of a single visit, the houses selected should be scattered throughout
the village. It is often advantageous to select the poorest and worst-ventilated
: 3
houses because they usually contain the largest numbers of mosquitos. Houses
on the fringe of a village or near known breeding sites will often yield more day-
1
2^1
resting mosquitos.
Mosquitos caught alive in houses may be kept for 24 hours. This will allow you
to check the 24-hour mortality rate among mosquitos collected from sprayed
houses and provide an indication of the care exercised when collections are
a
made in unprotected houses.
The collection of mosquitos in a house normally takes place early in the
morning and should be done after the occupants are up and dressed. Examine
the whole house or, if it is too large, spend up to 15 minutes searching in each
room. Pay special attention to those parts of the house most likely to yield
vector mosquitos, including rooms in which people slept the previous night,
the washing room, and any other areas suggested by experience. Try to select
rooms with few external openings. Mosquito catches tend to be highest on
mornings after heavy rain or when the humidity level is high.
Use of sucking tube
With the aid of the torch, look for mosquitos on walls, ceilings or the roof.
Search systematically, starting from the door and1 moving clockwise around the
inside of the house. Look for specimens on wal11 hangings and curtains, behind
and under furniture, and inside large pots and jars.
%
No more than five mosquitos should be collected in the sucking tube before
you transfer them to a paper cup.
%
Normal resting places of mosquitos in houses
While collecting, observe the resting places normally chosen by mosquitos. If
you record in your notebook what you see, including the numbers of the
insects on the upper, middle or lower parts of walls, you can determine the
proportions resting in each situation.
r.a
Labelling paper cups and making records of collections
During hand collecting you will need to transfer mosquitos to paper cups. You
should use a separate cup for each house. The cups must be clearly labelled in
pencil with at least the following essential information:
•
•
•
•
•
•
•
location
date and time of collection
time spent on collecting (minutes)
house number or householder's name
type of structure (house, animal shelter, store, etc.)
whether sprayed, and if so, when
your name.
Keep a separate record in your notebook of each collection you make.
35
If"
Entomological held techniques for malaria control: Learner s Guide
I
I
Preparation of collections and transport to the laboratory
The paper cups should be prepared for transportation as previously described.
A number of test-tubes with specimens from a single house can be held
together with a rubber band and transported in an insulated picnic box or other
container.
36
£
LEARNING UNIT 7
Spray sheet collection of mosquitos
1
j?
Learning objectives
I
I
'
I
By the end of this Unit you should be able to:
•
select a house and, with the permission of the householder, identify a suitable
room for spray sheet collection of mosquitos
•
prepare the selected room for spray sheet collection
•
carry out space spraying with a pyrethrin solution
•
collect all the mosquitos that have fallen
•
label containers accurately and completely, record results, and transport mosqui
tos to the laboratory.
, I
T
:P
if
if8
Spray sheet collection involves the use of a pyrethrin space spray to knock
down mosquitos resting inside a house and collecting them on white sheets
spread on all flat surfaces. The mosquitos are transported to a laboratory for
processing.
The hand collection of mosquitos that you carried out in Learning Unit 6 is
unlikely to result in your obtaining all the mosquitos resting in a house, for the
following reasons:
•
•
•
4
many mosquitos may be hidden from sight, particularly in houses with
thatched roofs;
roofs, ceilings and the upper parts of walls may be too high for you to reach;
you may not have sufficient time available to collect all the mosquitos.
■3
3
The efficiency of hand collecting also varies with the absolute density of
mosquitos. It is therefore unwise to use data from hand collections for
mathematical calculations.
3
Using the sheet collection method, it should be possible to collect practically all
the mosquitos from a well-closed room sprayed with a fine mist of 0.2-0.3%
pyrethrin solution. Even in a less well-closed room you will be able to collect a
high proportion of the mosquitos resting there.
This method of collection allows quantitative studies to be undertaken,
including measurement of:
•
■R
•
37
indoor resting density (the number of mosquitos resting indoors during the
day)
seasonal changes in indoor resting density
Entomological held techniques for malaria control. Learner 's Guide
the number of mosquitos remaining in a particular room after a hand
•
collection.
On arrival in the laboratory, the mosquitos are examined and dissected to
determine:
•
their blood digestion/egg development stages
•
•
•
their ages
the proportion having sporozoites in their salivary glands
the proportion that have fed on human blood.
Although spray sheet collecting was usually widely accepted in the past, you
may encounter some resistance from householders. The physical appearance of
the collecting sheets deteriorates markedly after they have been used only once
or twice and some people may be reluctant to have them in their houses,
especially in areas where housing standards are relatively sophisticated.
Essential equipment
To carry out pyrethrin spray sheet collection you will need the following
equipment: white floor sheets; hand sprayers; a hand lens; pyrethrin solution;
small Petri dishes; pill boxes, paper cups or metal boxes; hand lens; forceps; a
container, preferably a picnic box, for transporting mosquitos to the laboratory;
cotton wool; filter paper; a torch (with spare bulb and batteries); a pencil; and a
notebook.
The white sheets (sizes 2 m x 2 m and 2 m x 1 m) can be made of cotton; it is
often useful to have one or two larger sheets. The number of sheets required
depends on the average size of the rooms.
The hand sprayers should be of the double-action type with an air valve (see
Fig. 10). Pyrethrin solution, at a concentration of 0.2-0.3% in kerosene, is
normally available ready-mixed from your store in quantities sufficient for
several weeks' work. If new stocks are required you should prepare them only
under the guidance and supervision of a person familiar with the methods used.
Fig 10
Hand sprayer
If there is likely to be a delay between collection and delivery to the laboratory,
wide-mouthed vacuum flasks or an insulated picnic box should be used so that
the Petri dishes or other containers for the mosquitos can be kept cool over ice
bags.
38
s
: -s
1J
r’
Spray sheet collection ol mosquitos
Selection of rooms for spray sheet collection
Collections are normally made in the morning after the occupants of houses
have risen and dressed. Houses will already have been selected if the work is to
be done in a locality where regular observations are made; otherwise, selection
is a responsibility of the supervisor. In principle, the houses chosen should be
distributed throughout the locality.
Permission is required from householders to make collections in their houses.
It is normal for the work to be performed by a team of three or four people so
that collections can be made in eight to ten rooms in each locality.
-i*
*
Preparation of rooms for spray sheet collection
Ensuring that you disturb any resting mosquitos as little as possible, prepare a
room for spraying as follows:
•
•
•
•
: %
Remove all animals and birds.
Remove or cover all food.
Remove all small items of furniture.
Cover all openings and eaves with cloth or mosquito netting.
white sheets
c
%
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'///////////////^^^
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mosquito
netting or cloth
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1white sheets
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Fig 11. Preparation of a room for pyrethrin spraying
39
ft
I
Entomological field techniques for malaria control. Learner s Guide
. Spread the white sheets so that they completely cover the floor and all flat
surfaces of the remaining furniture. Sheets should also be spread under
tables, beds and other places where mosquitos may hide. Sheets placed on
furniture should not hang down to the floor because this would prevent the
spray from reaching mosquitos under the furniture (see Fig. 11).
• Close all windows and doors.
Carrying out space spraying
One of the team members should walk round the outside of the room and spray
in open spaces or holes in the walls and eaves. The same person or another
member of the team should then enter the room, close the door and, moving in a
is ----filled-----with
clockwise direction, apply spray towards the ceiling until the room u
the
size
of
the
a fine mist. The time needed for spraying obviously depends on
that
the
door
room. The operator should leave the room quickly and make sure
remains closed for at least 10 minutes. Great care should be taken to avoid
treading on any mosquitos that fall early. Many workers remove their footwear
to avoid contamination of the white sheets.
Collection of mosquitos from sheets
Two methods can be used to collect mosquitos from the sheets following space
spraying with pyrethrin. Each involves picking up the mosquitos with forceps
and placing them in a container for transport to the laboratory. To prevent
mosquitos from drying out if they are to be used for dissection, there should be
a layer of damp cotton wool covered by filter paper in the container.
Method 1
Open the room. Move gradually through the room, starting from the
doorway. With the aid of a torch, collect the mosquitos from the sheets; leave
the sheets in position.
Method 2
Open the room. Starting from the doorway, pick up the sheets one at a
time by their corners. Carry the sheets outside. Collect and examine the
mosquitos outside in daylight.
If it is raining or windy, the first method should be used. Collect up and fold the
sheets; ensure that you leave no dust or debris behind.
Labelling containers and transporting them to the laboratory
A separate container should be used for the mosquitos collected in each room.
The containers should be labelled with all relevant data, including.
•
•
•
•
•
40
location
date and time of collection
house number or householder's name
type of structure (house, animal shelter, store, etc.)
number of people and/or animals in the room during the previous night
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Spta/ sheet collection of mosquitos
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• whether the room has been sprayed previously and, if so, the date of
spraying
• name of collector or supervisor.
This information must be written in pencil directly on paper cups or on slips of
paper which are placed inside other types of container.
r
- es
If the mosquitos that have been collected are to be transported rapidly to the
laboratory, the containers can be placed in a box for the journey. However, if
there is to be any delay before transport, a wide-mouthed vacuum flask holding
ice should be used to keep the containers cool so that the mosquitos remain in a
suitable condition for dissection; alternatively, an insulated picnic box with ice
packs may be used if one is available.
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LEARNING UNIT 8
Outdoor collection of mosquitos
;-r«
Learning objectives
By the end of this Unit you should be able to:
,
• describe the sites and methods used to collect resting adult anopheline mosquitos
from natural outdoor locations
i
• collect outdoor-resting mosquitos using a sucking tube
r
r
u
• collect outdoor-resting mosquitos using a drop net
• collect outdoor-resting mosquitos using a hand net
• construct artificial shelters and collect anopheline mosquitos from them
• label paper cups completely and accurately, maintain records of collections, and
transport mosquitos to the laboratory.
Some mosquito species enter houses at night to bite and rest indoors during the
day. Other species do not enter buildings but bite outside and then rest in the
following kinds of outdoor location:
•
•
A
A
? %
on vegetation;
on solid surfaces in sheltered places, such as the banks of streams and ditches,
holes in rocks, culverts, cracks in stone walls, caves, animal burrows, on the
trunks or stems of larger vegetation such as banana trees, and in old termite
mounds.
Outdoor collecting is performed in either the natural resting places described
above or in shelters specially constructed for the purpose. Artificial shelters
have the advantage of providing concentrated sites for collections and more
representative samples that can be used for quantitative work.
*
7•&
Data from outdoor collections are important in evaluating the impact of any
anti-vector measures, and provide information about:
•
the species that habitually rest outdoors
•
•
•
the relative numbers of mosquitos resting outdoors
seasonal changes in outdoor resting habits
any alterations in the relative numbers of mosquitos resting outdoors
following the application of insecticides in houses and other buildings.
Smears made on filter paper from blood-fed specimens may be subjected to
precipitin tests to determine the host preferences of mosquitos.
43
Entomological field techniques for malaria control: Learners Guide
Essential equipment
The equipment required for outdoor collecting consists of: a sucking tube, a
torch (with spare bulb and batteries); a hand net; a drop net; paper cups with net
covers; cotton wool; an insulated picnic box; a towel; newspapers for use as
packing material; a pencil; and a notebook.
Since the preparation or construction of artificial shelters will be undertaken
during field practice, you also require: a barrel; two spades; a pickaxe; and an
axe or machete.
Choice of method for outdoor collecting
The choice of method for outdoor collecting depends partly on the behaviour
of the malaria vector; that is, whether it prefers to rest on vegetation or on solid
surfaces.
" ; on vegetation
The three common methods used to collect mosquitos resting
net, and a drop net.
involve the use of a sucking tube and torch, a hand
1
Anopheline species that normally rest on solid surfaces are collected with the
aid of a sucking tube and torch from natural or artificial shelters. Both pwticuvC
and skill are essential in collecting from natural shelters, since the population
density of mosquitos is usually much lower than in artificial shelters and the
resting surfaces are irregular.
Collecting mosquitos outdoors with a sucking tube
Direct collection from vegetation using a sucking tube (and transferring
captured mosquitos to a paper cup) usually takes a considerable time and may
enable you to find only a few mosquitos. Collections from both natural and
artificial shelters are also made using a sucking tube and torch. Well-placed
shelters normally yield more mosquitos than natural environments.
There is an important difference between these two kinds of collecting in
respect of the information that should be recorded:
•
After searches in vegetation you should record the number of collections
•
and the total time spent searching.
After searches in artificial shelters you should record the number of shelters
examined and the time spent in each.
Collecting mosquitos outdoors using a hand net
A hand net (or sweep net) like that illustrated in Fig. 12 is used to collect
mosquitos resting on vegetation. The correct method of use is to move the
hand net swiftly over the tops of tall grasses or close to the ground around
bushes.
Make sure that you record the number of collections and the total time spent
collecting.
44
Outdoor collection of mosquitos
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Fig 12 Hand net
Collecting mosquitos outdoors using a drop net
A drop net (Fig. 13) consists of a light, collapsible wooden frame measuring
about 2 m x 2 m x 2 m, with its top and sides covered by a mosquito net.
The drop net is placed over grass or low vegetation. Any mosquitos beneath it
are disturbed with a stick so that they fly up and rest on the net. The mosquitos
are collected from inside the net with a sucking tube and transferred to a paper
cup.
t !
■La
Record the number of times the drop net is positioned for collecting; this
indicates the area that has been searched.
Collecting mosquitos from artificial outdoor shelters
If the anopheline vectors prefer to rest on solid surfaces outdoors, it is preferable
to build shelters to .attract them. Artificial shelters are easier to search than
natural resting places; they may consist of large barrels or boxes, perhaps set
into river banks, or they may be pits dug in the ground (see Fig. 14). In general,
however, artificial shelters of this type are used only in locations where regular
mosquito collections can be made and should be sited where they are unlikely to
be flooded in the wet season.
Collections from outdoor shelters are made using a sucking tube and a torch.
Before entering any natural or artificial shelter, it is important to make sure that
it contains no snakes or other small wild animals.
4
The number of shelters that have been searched must be recorded.
^•3
Labelling and recording collections
All paper cup containers must be labelled in pencil with the following
-5 Obj
information:
45
I
Entomological field techniques tor malaria control. Learner s Guide
hl
i
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___
Net being lowered
Net in place
Fig. 13
•
•
•
•
Drop net for collecting mosquitos from grass
location
method of collection
date and time of collection
whether the nearest village was treated with insecticide, and when
• the collector's name.
The data derived from outdoor collections are used to calculate the following
indices:
•
number of mosquitos collected per person-hour, based on outdoor collection with a sucking tube and/or a hand net;
number of mosquitos collected per unit area, based on outdoor collection
•
with a drop net;
number of mosquitos collected per artificial shelter, based on outdoor
•
collection from such shelters.
46
J
Outdoor collection of mosquitos
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Pit shelter with roof (top view)
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Barrel as resting place covered with earth and branches
Fig 14 Artificial outdoor shelters
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47
: a
LEARNING UNIT 9
Direct catches of mosquitos from bait
I
Learning objectives
By the end of this Unit you should be able to:
•
i
describe when, where and how to make direct collections of mosquitos from
human bait
•
perform direct-biting collections from human bait
•
describe when, where and how to make direct-biting collections of mosquitos from
animal bait
•
perform direct-biting collections of mosquitos from animals
•
maintain, and completely and accurately label and record, collections from the
field
•
prepare collections for transport to the laboratory.
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Female mosquitos are attracted to humans and/or animals to obtain blood
meals. The number of vectors biting humans is therefore a major determinant of
malaria transmission, and it is important to know:
•
•
•
•
•
•
•
which anopheline mosquitos bite humans
which of the anophelines that bite humans are vectors of malaria
how often a person is bitten by a vector
the time of day when vectors bite
the peak biting time
whether the vectors bite indoors or outdoors
the seasonal variations in the numbers of mosquitos biting humans.
Following the start of a malaria control operation it is also necessary to
determine whether there are changes in:
•
•
•
the anopheline species biting humans
the numbers of vectors biting humans
the numbers of vectors biting humans indoors compared with the numbers
doing so outdoors.
Many vectors bite both humans and animals. Collections should therefore be
made on animals to discover whether the vector:
'•3
•
•
•
49
bites only humans
prefers humans but also bites animals
prefers animals but occasionally bites humans.
Entomological field techniques for malaria control: Learner's Guide
Essential equipment
In order to capture mosquitos as they are biting you need the following
equipment: a sucking tube; a test-tube; a torch (with spare bulb and batteries);
paper cups with net covers; rope (for tethering the animal bait); long metal or
wooden pegs (to which to tie the tether); hammer; cotton wool; towels; an
insulated picnic box with packing material; a pencil; and a notebook.
General rules
Certain important rules should be observed while carrying out this type of
collection:
•
•
Do not smoke while collecting.
Change the people being used as human bait hourly, so as to minimize
•
possible differences in their attractiveness to mosquitos.
Do not use any oil or ointment that might act as a mosquito repellent.
Siting of bait
Human bait indoors
If possible, select a house in that area of the village with the greatest number of
cases of malaria. The house should be typical of the locality.
A house with more than one room is preferred, allowing the usual residents to
sleep in one room while another is used by the collector(s). If possible, the room
used for collecting should be one in which the residents would normally sleep.
Unless it is raining, collections should not be made on an open verandah, which
is considered to be neither inside nor outside the house.
Human bait outdoors
The person acting as human bait for outdoor collecting should be positioned in
the general area of the house or room selected for indoor collecting. The site
selected should be an area of the village where local people normally sit during
the evening.
Animal bait
It is usual for collections from animals to be made outdoors, even in areas
where livestock is normally stabled. It is difficult and dangerous to collect
directly from a large animal in an enclosed space, especially when other animals
are present.
The animal chosen should be stationed a short distance from other animals. It
can then be assumed that blood-fed mosquitos resting; on nearby vegetation
have taken blood from the bait animal, and these can <also be collected.
Efficiency of direct collecting
Collections taken directly from human bait using sucking tubes normally reflect
quite accurately the number of mosquitos biting. Collections made using trap
nets with human bait generally yield fewer mosquitos and less representative
samples.
50
Direct catches of mosquitos from bait
•
T
c
Direct collecting from large animals is very difficult to carry out efficiently and
only a small proportion of the mosquitos biting will be obtained. Much greater
numbers of mosquitos are usually captured by using a trap net baited with an
animal.
When and where to make direct catches from human bait
Direct collecting of biting mosquitos is performed during the night because this
is when most malaria vectors take blood meals. The collections are often made
during the entire period from dusk to dawn. However, this should be done only
when absolutely necessary, and then by two teams of collectors, each working
half the night. If the peak biting time is known, collecting can be confined to
this period. Peak biting times can be deduced early in a programme from the
results of several all-night collections, allowing subsequent regular collections
to be made over a period of only three to four hours. Thus, both indoor and
outdoor collecting from human bait may be conducted to accommodate the
normal resting and sleeping habits of the local people. Remember that night
time habits may vary seasonally.
1 :S
4 ; s!
The times and locations for night collecting will be selected by the supervisor.
It is important that you do not smoke during collections and that you keep as
still as possible when collecting mosquitos from your own body.
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Direct collecting of biting mosquitos from human bait
The collection of mosquitos from your own body is a common way of
obtaining biting specimens, especially of Anopheles. The technique is as follows
(see Fig. 15):
•
Select a quiet place either inside or outside a house as instructed by a
supervisor.
F 3
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55
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55
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Fig 15. Collecting from your own body
51
LIBRARY “X
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DOCUMENTATION
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Entomological held techniques for malaria control. Learner's Guide
• Adjust your clothing so that your legs are exposed as far as your knees.
Then sit quietly.
• When you feel a bite, quickly turn on your torch to find out whether the
mosquito is anopheline or culicine.
• If the mosquito is anopheline, or- if you are unsure, collect it with your
Use one cup for each hour of
sucking tube and transfer it to a container.
(
collecting.
• Do not smoke or move around.
Alternatively, one person can serve as bait and another as collector (see Fig.
16). In this case, the technique is as follows:
• The person acting as bait sits or lies in a quiet place, inside or outside the
house as appropriate, with his or her clothing adjusted to expose as much
skin as is acceptable.
• Using a torch and a sucking tube or test-tube, the collector checks for and
collects biting anophelines every two> or three minutes.
to a paper cup. One cup is used for each hour
• The mosquitos are transferred
1
of collecting.
Fig 16 Collecting from another person’s body
Ethical considerations
In the past it was considered acceptable to allow mosquitos to bite a person
engaged in night collecting. However, for ethical reasons it is now desirable to
examine whether health workers should be routinely exposed to the risk of
contracting malaria. In fact, it is not necessary to permit mosquitos to feed: they
can be collected as soon as they settle on the skin, since it can be safely
assumed that biting would normally follow. Landing rates should therefore be
measured instead of biting rates.
Nevertheless, those involved in collecting will continue to be bitten; since
mosquito repellents cannot be used, this is unavoidable. It is, of course,
understood that both collectors and people serving as bait should take an
appropriate drug for prophylaxis of malaria.
When trap nets are used, the risk to collectors can be minimized by using
protective inner nets (see Fig. 17), although this reduces the efficiency of
collection.
52
Direct cutches of mosquitos from hail
When and where to collect from animal bait
i*
if
F
Many malaria vectors are■ attracted in large numbers to animals, and collecting
mosquitos on or around an
< animal can therefore be highly productive.
Collecting from animal bait is normally carried out in the same location and at
the same time as collecting from human bait. The exact period of collection is
chosen by the supervisor.
Before sunset, select a tame animal from the village, usually a cow or water
buffalo. Negotiate with the owner and explain how the animal is going to be
used. The collecting site should be near the place where the animal usually
passes the night. Then proceed as follows:
• Before sunset, tie the animal securely using a short tether attached to
wooden or metal pegs driven firmly into the ground.
• At sunset begin collecting mosquitos by means of a torch and sucking tube.
• Examine the animal every two to three minutes and collect all the
anopheline mosquitos you find.
• Keep each hour's collection in a separate paper cup.
Labelling, recording and preserving hourly collections
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The paper cups containing the hourly collections must be very clearly labelled
to ensure that no confusion arises. The information to be recorded on the cups
is as follows:
•
•
•
•
•
•
•
location
date
whether and when the location was last sprayed
type of bait (human or animal)
site of collection (indoors or outdoors)
hour of collection (e.g., 18 h to 19 h)
the collector's name.
Keep the paper cups in an insulated picnic box which, when closed, will not
allow ants to get in. The humidity in the cups should be maintained by means
of damp cotton wool pads.
3
Transportation to laboratory
Collections should be transported to the laboratory in the picnic box. Take all
the necessary precautions you have already learnt about.
*
53
i '
LEARNING UNIT 10
'I*
1
Collecting mosquitos in baited
trap nets
Learning objectives
By the end of this Unit you should be able to:
• explain when collections should be made from human-baited trap nets
• collect mosquitos in a human-baited trap net
• explain where collections from animal-baited trap nets should be made
• collect mosquitos in an animal-baited trap net
-
• maintain, and completely and accurately label and record, trap net collections
' ■ S3
• prepare collections for transportation to the laboratory.
' I ,!S
:1 -s
I
I__________ _____________________________
Collecting mosquitos from human and animal baits necessarily involves a large
number of workers if good results are to be obtained. Methods have therefore
been developed that use trap nets with either human or animal bait. These
require fewer collections and therefore allow the workers periods of sleep
during a night's work. These alternative methods also provide valuable
information about the numbers of mosquitos biting humans and animals, and
are thus often used in place of direct collections.
Animal-baited trap nets generally produce more mosquitos than can be
collected by direct capture from animals; the opposite, however, is true for
human-baited trap nets. For this reason, standard night collecting from bait
usually involves direct collection from humans indoors and outdoors, and
collection from animal-baited trap nets outdoors.
X
In this Unit you will learn how to use trap nets; the purposes of collection by
this method are the same as those of direct collecting, noted earlier.
Essential equipment
1*5
iS
You will require the following equipment in order to set up trap nets for two
human baits (indoors and outdoors) and one animal bait (outdoors): a sucking
tube; a torch (with spare bulb and batteries); paper cups with net covers; cotton
wool; towels; an insulated picnic box; a pencil; a notebook; an alarm clock; two
camp beds; two small mosquito nets with frames to fit the camp beds; two trap
nets for human bait; one trap net for animal bait; long metal or wooden pegs and
a rope (for tethering the animal); hammer; pegs and string (for securing trap
nets); and a needle and thread (for repairing trap nets).
55
Entomological field techniques for malaria control. Learner s Gi/'de
General rules
The following important rules should be observed:
• Do not smoke while collecting.
• People acting as bait indoors should change with those acting as outdoor
bait on alternate collecting nights; this allows for differences in attract
iveness to mosquitos.
• Keep collections separate according to bait, site and collecting period, and
label them correctly.
Collection sites
Two trap nets will be set up in positions selected by the supervisor, one in a
sleeping room and the second outdoors at a site where people habitually sit
during the evening or sleep.
Collecting by means of human-baited trap nets
A human-baited trap net is set up as shown in Fig. 17 on a site either inside or
outside a house. The procedure is as follows:
Set up a folding camp-bed.
Put up the inner net around the bed to protect the person acting as bait.
Erect the outer net by securely tying it to poles or branches.
Stretch the bottom of the outer net tightly and tie it to pegs in the ground,
leaving 15-20 cm between the ground and the lower edge of the net.
• At sunset, get into the trap and lie on the bed or, if someone else is acting as
the bait, ask him or her to do so.
• Set the alarm clock to ring after one hour during a season of high mosquito
density, two hours in a moderale-density season, or three hours in a lowdensity season. The collecting period should not exceed ten minutes.
• When the alarm rings, collect all the anophelines in the trap net.
•
•
•
•
n
I
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Fiq. 17. Trap net with human as bait
56
Collecting mosquitos in baited trap nets
&
•
•
i?
iJ
•
Transfer the mosquitos to a container and label it with the date, time,
location, and your name. Use one container for each collecting period.
Get back onto the bed and set the alarm to ring after the same length of time
as before.
Repeat the procedure throughout the night.
If the protective inner bed net is not used, and the mosquitos are thus allowed to
feed on the bait, collecting may be done over longer periods, depending on the
objective. This is because fully fed mosquitos will be more settled and will rest
longer on the sides of the trap net than those that have not fed.
Siting of an animal-baited trap net
An animal-baited trap net is sited close to where the animal is customarily kept
overnight.
I
Animal-baited traps are normally used outdoors only. It is difficult to confine a
cow or buffalo in a net in a stable where there are other animals; also, use of this
method indoors frequently results in damage to the net.
Collecting by means of animal-baited trap nets
The trap net (see Fig. 18) is similar to that used for collecting mosquitos
attracted to human bait. The animal must be securely tethered so that it cannot
break free and damage the trap or harm itself. If there is to be repeated collecting
at the site, a small enclosure may be built to confine the animal, as shown in
Fig. 18.
:: sc
; 4
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Fig 18
Animal-baited trap net
The animal is placed in the trap at sunset and mosquitos are collected every
three hours, for instance at 21 h, 24 h, 03 h, and sunrise. A single collection
obtained at dawn produces only about a third of the mosquitos that can be
caught if collecting is done every three hours.
57
Entomological he'd techniques lor malaria control Learner's Guide
Make sure that all containers are labelled correctly. Because of the large
numbers of mosquitos obtained when animal baits are used, it may be
necessary to use several paper cups during each collection. Do not put more
than 50 mosquitos in each cup.
Using animals is a good way to collect large numbers of mosquitos but it does
not give accurate information about the times at which biting occurs. This
information is best obtained by making hourly collections during the night.
Preserving, labelling, recording and transporting collections
All the procedures described in Learning Unit 9 for direct collecting of biting
mosquitos apply equally to collections from baited trap nets.
Note: Nets should be thoroughly dried in the sun, either in the field or on return
to the laboratory, before being packed away.
58
1,
LEARNING UNIT 11
1
1
I
Collecting mosquitos from exit traps
i*
Learning objectives
By the end of this Unit you should be able to:
•
fit exit traps to houses and other buildings
• describe the precautions necessary for the correct operation of exit traps
• collect mosquitos from exit traps at appropriate times
•
: !-s
label and prepare specimens for transport to the laboratory.
Exit traps (see Fig. 19), which'i can be fitted to houses and animal shelters, are
normally use<d only for certain special purposes, such as observing the daily
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B. Pattern for cutting mosquito netting, excluding sleeve side
Fig. 19. Exit trap
59
Entomological held techniques for malaria control: Learners Guide
movements of mosquitos into and out of houses, and distinguishing between
the mosquito species that both bite and rest indoors and those that bite indoors
but rest outdoors. Studies carried out before and after indoor spraying can also
be used to assess the following:
• the effects of insecticides on the normal movement and feeding habits of
mosquitos;
J.j as indicated by the numbers of dead
• the residual effects of insecticides
,d
by
the
24-hour mortality rate of mosquitos found
mosquitos collected am
alive in the traps.
Exit traps are suitable for fitting only to rooms that are well sealed and that
have few exit points for mosquitos (see Fig. 20). Collecting by this means must
be done under the strict supervision of an experienced entomologist.
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Fig 20. Exit traps fitted to door and window
60
Collecting mosquitos from exit traps
k
Essential equipment
Since the type and positioning of exit traps will vary according to the design of
houses and
and animal
animal shelters
particular areas, it is not possible to list the
houses
shelters in
in particular
materials necessary for fixing traps. However, the other equipment you will
need is as follows: a sucking tube; paper cups with net covers; towels; an
insulated picnic box; dark cloth or netting to block openings in rooms; and the
exit traps themselves.
Fitting exit traps
I
ri
The selection of houses and/or animal shelters on which to fit exit traps is
important. Those with few openings are the most suitable since efforts must be
made to cover or block with dark cloth all openings other than the windows or
doors to which exit traps are to be fitted. The mosquitos that enter an exit trap
are attracted to the faint light that comes through the trap opening.
b
It is also important to fix traps in position well before sunset. Collecting should
then cover a period of two to three hours after sunset; this will reduce mortality
due to nocturnal climatic factors (wind, rain, cold).
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One room, normally a sleeping room, is selected and the trap is fitted to a
window. The method of fitting depends on the type of house and the window,
and also varies according to whether there will be a single collection or regular
collections. For regular collections, a permanent arrangement for fixing the trap
can be considered, and any parts of the window not covered by the trap can be
covered with dark cloth or hardboard.
The trap is fitted to the window with the collecting sleeve outwards. All other
large openings, including eaves, have to be covered with dark cloth. A few
small openings must remain to allow the entry of mosquitos.
In areas where houses or huts do not have windows, the exit trap may be fixed
to the door. In this event, it is constructed with a light wire frame and attached
to a piece of thick dark cloth which is longer and wider than the door-frame.
The top and one side of the cloth are attached to the door-frame or wall, while
the other side and the bottom remain unattached so that people can still enter
or leave the house.
Precautions ensuring correct operation of exit traps
t*s
The supervisor will explain to the occupants of the chosen house that people
should continue to sleep in rooms with traps and that windows to which traps
are fitted must remain open. You must check that these windows have been left
open when you go to collect mosquitos.
9
Where traps are attached to doors you should verify, when making collections,
that the doors are open and that the cloth is preventing the exit of mosquitos
(i.e. there should be no openings through which mosquitos may escape).
If you believe that the occupants may have used an aerosol spray or pyrethrin
coil for mosquito control, inform your supervisor.
61
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LEARNING UNIT 12
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Collecting larvae and pupae from
breeding sites
Learning objectives
By the end of this Unit you should be able to:
•
describe the most likely breeding sites of malaria vectors
•
describe the methods of collecting mosquito larvae and pupae and demonstrate
skill in using a dipper, a larval net, a well net, and a pipette or spoon
•
maintain accurate records of all collections of larvae and pupae
•
transport live larvae and pupae collected in the field to the laboratory
•
kill and preserve larval and pupal specimens.
Each type of mosquito prefers to lay its eggs in a particular kind of water. Some
will lay only in fresh, clear, running water with some shade, others only in
brackish water; some may even lay eggs in very small quantities of water, such
as that in a discarded tin can or trapped in a hoof-print.
■L
It is most important for you to know the preferred breeding sites of the
anopheline mosquitos that transmit malaria, and the densities of larvae and
pupae at these sites. The methods by which you will collect larvae and pupae
will depend on these variables.
Collecting from all the different types of breeding site in an area will allow you
to:
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•
•
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determine the species present
determine the preferred breeding sites of each vector species
determine whether larvae and pupae are present during a control pro
gramme directed against adult mosquitos even though these may be rare or
uncollectable
make an assessment of the effectiveness of a programme directed against
larvae and pupae.
Essential equipment
■
«
The equipment required for the various methods of collecting larvae consists
of: a dipper; a larval net; a well net; a spoon; a large tray; a pipette; stoppered
specimen tubes (vials); 70% alcohol solution or 2% formalin solution; a pencil;
and a notebook.
If live specimens are required for insecticide testing you will also need larger
bottles or a wide-mouthed vacuum flask.
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63
Entomological field techniques for malana control L earner s Guide
Preferred breeding sites of anopheline mosquitos
To identify the preferred breeding sites, it is essential to be systematic and
check all possible breeding places, even those that are hard to reach. This
enables you to determine the types of site most likely to harbour the larvae of
anopheline mosquitos.
Potential breeding sites include:
•
small pools, tin cans, hoof-prints, drains and ditches, where the entire surface
•
•
of the water should be examined;
brackish water, e.g. at sites where fresh water and salt water mix;
streams, which should be searched at the edges where there is vegetation
•
and the water moves slowly;
ponds and lakes, where larvae usually occur in vegetation around the edges
•
but can sometimes be found far from the shore among floating vegetation;
swamps and marshes, where larvae occur in places similar to those described
•
for ponds and lakes;
special sites, such as wells and water containers made of cement, where the
entire surface of the w^ater should be examined.
Anopheline larvae and pupae are often concentrated in certain parts of large
breeding sites. If these locations are known, they can be used regularly for
collecting.
Whichever collecting method is used, you must always approach the breeding
place cautiously, facing the sun: if the larvae are disturbed by shadows and
movement many of them will swim downwards and disappear from view. You
will then have to wait quietly for several minutes until they return to the
surface of the water before you can collect your samples.
Methods for collecting larvae and pupae
i
Use of the dipper
Various kinds of dipper are used, including small frying pans, soup ladles and
photographic dishes (Fig. 21); it is important to use the right type and size for
each breeding place. A white enamelled dipper is preferable, because this allows
you to see the larvae most easily.
The method of use (see Fig. 22) is as follows.
•
•
•
Lower the dipper gently into the water at an angle of about 45 , until one
side is just below the surface.
While dipping, care should be taken not to disturb the larvae and thus cause
them to swim downwards. If they are disturbed, wait for a minute or two
until they come up to the surface again, and then continue dipping.
Move along the breeding site, skimming the surface of the water with the
dipper.
Lift the dipper out of the water, making sure that you do not spill the water
containing the larvae and pupae.
• Hold the dipper steady until the larvae and pupae rise to the surface of the
•
water.
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64
Collecting larvae one pupae from breeding sites
Fig 21. Types of larval dipper
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Fig. 22. Using a larval dipper
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• Collect the larvae and pupae by means of a pipette and transfer them to a
bottle or vial.
• Do not throw the residual water back into the breeding place, as this may
further disturb the larvae and pupae.
An alternative method is to hold the dipper at an angle of 45° and lower it
gently until it is just below the surface so that water flows in with any larvae
and pupae that may be present.
Where there is dense, floating vegetation or debris, use the following method:
• Disturb the water, causing the larvae and pupae to sink below the surface.
• Clear away the vegetation or debris with the dipper and wait a few minutes
for the larvae and pupae to return to the surface.
• Collect the larvae and pupae with the dipper as described above.
Where there are clumps of vegetation such as tall grasses, press the dipper into
the clump and lower it into the water so that the surface water flows in. Then
take the dipper out of the water and collect larvae and pupae as indicated
above.
65
Entomological field techniques for malaria control: Learner's Guide
Use of the larval net
A larval net for collecting larvae and pupae in ponds and lakes consists of a fine
mesh net which has a plastic bottle or tube tied to one end and is mounted on a
wooden handle (see Fig. 23). To collect larvae and pupae, sweep the water
surface by holding the net at an angle and moving it through the water (see Fig.
24). Larvae and pupae on the water surface will be swept into the net and will
collect in the plastic bottle or tube.
Alternatively, a simple net with no attached bottle or tube can be used. After
sweeping, the net should be inverted into a bowl of water and its contents
dislodged. The water in the bowl is then searched for larvae and pupae, which
are picked up and transferred to a bottle or vial by means of a pipette.
netting
plastic
tube
Fig. 23. Larval net
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Fig 24 Using a larval net
66
Wrong way
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Collecting larvae and pupae from breeding sites
Use of the well net
The net used for sampling from wells is similar to the larval net but lacks the
wooden handle; instead, it is held at an angle by four strings and controlled by
a long string or rope (see Fig. 25). The method of use is as follows:
•
•
•
Lower the net into the well so that the lower side of the net is just under the
surface of the water and its opening is at an angle of about 45° (see Fig. 26).
Move the net right round the side of the well once or twice.
Withdraw the net and look for larvae and pupae in the bottle or tube at the
end of the net.
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Fig. 25. Well net
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67
Using a well net
Entomological held techniques for malaria control. Learners Guide
Using a small spoon or pipette
A small spoon or wide-mouth
wide-mouth pipette
pipette can be used for collecting larvae and
pupae from small amounts of water, e.g. in a tin can or trapped in a hoof-print.
The method is as follows:
• Stir the water vigorously with a stick to make it muddy.
• Watch for the larvae and pupae to rise to the water surface: they are easily
seen against the muddy background.
• Pick up the larvae and pupae with the spoon or pipette.
After collecting by this or any of the above methods, you should transfer the
larvae and pupae to specimen bottles or vials.
Maintaining accurate records of collections
Draw a sketch-map to show the positions of the oreeoing sites relative to
villages and settlements. As you collect you should give each breeding place a
number in your notebook. Against this number, record the location, type of
breeding place, number of dips made or time spent sampling, and the date.
Place all the specimens from a particular breeding place in one bottle or vial and
label it with the number recorded in your notebook. The number must be
written in pencil on a piece of paper and dropped into the specimen bottle. Do
not use a ballpoint pen as the ink dissolves in water.
Transporting live larvae and pupae to the laboratory
The larvae and pupae collected must arrive alive and undamaged at the
laboratory.
Stopper each bottle or vial tightly so that water cannot spill. Make sure that
there is air in the top 1-2 cm so that the larvae and pupae can breathe for a few
hours. If a larger air space is left, the water will become agitated during
transportation and the specimens will suffer damage, particularly loss of hairs. If
the journey to the laboratory takes more than two to three hours, remove the
stoppers every two hours to provide the specimens with fresh air.
Pack bottles and vials carefully so that they are not jolted during transportation.
If the larvae are to be used in insecticide susceptibility tests they should be
transported in water in a large vacuum flask or other large container.
Killing and preserving larvae and pupae
If the laboratory asks you to provide preserved larvae and pupae:
• Kill them by placing them in warm water i.e., at 60 C.
• Remove the larvae and pupae from the water and put them into specimen
tubes containing 70% alcohol.
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68
Collecting larvae and pupae from breeding sites
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• Put a label on each tube showing the collection number.
• Close the tubes tightly.
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Another commonly used method of killing and preserving larvae and pupae in
the field is to add a few drops of 2% formalin solution to vials containing
freshly collected larvae or, preferably, to place the larvae in vials containing
2% formalin solution.
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LEARNING UNIT 13
Assisting the field supervisor
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Learning objectives
By the end of this Unit you should be able to:
• describe what the supervisor expects from you in order to prepare complete and
accurate records of your work and maintain equipment in good working order
• describe what kind of information on malaria you are now able to provide to the
community.
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The field supervisor is responsible for collecting and recording information on
project record forms. You can help with this task by ensuring that you carry
out all collections correctly and by labelling the paper cups or other containers
with all the required information.
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It is important that you inform the supervisor of any unusual circumstances that
might have affected your collections, such as there being no mosquitos in an
exit trap because someone closed the window to which it was fixed, or the use
by a householder of an aerosol insecticide or mosquito coil.
Maintenance of equipment
The supervisor is resj;ponsible for ensuring that the correct collecting equipment
is brought to the fieldd in good condition. The workers collecting mosquitos are
individually res;ponsible for the equipment issued to them.
*
Collectors are iusually also responsible for the packing of equipment after use.
ou must inform the supervisor of any loss; or damage to equipment, such as
splitting or holing
1
of trap nets and exit traps, or breakage of test-tubes and
sucking tubes,, so that remedial action can be taken before the next field
operation.
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Giving information to the community
In a malaria control programme, certain people are usually responsible for
providing information on malaria and'or genera! health education to local
communities. Their visits to any particular community do not occur very often,
whereas yours will be more frequent. During your visits it is quite likely that
people will ask you questions about malaria, mosquitos and the reasons for
your work. The information you have received during training should enable
you to answer most such questions.
Remember: When answering questions, only give replies that you know are
correct. If you cannot answer a question, pass it on to your supervisor.
I
71
I
Explanation of terms
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Explanation of terms used in the manual
adult
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The adult (imago) anopheline mosquito is a slender,
delicate insect with comparatively long, thin legs.
The outer covering of the body is composed of a
tough substance called chitin. The body is divided
into three distinct parts: head, thorax and abdomen.
anopheline mos<iquitos Members of the group of mosquitos having the
scientific name Anopheles. Only certain species of
anopheline mosquito carry human malaria.
Arthropoda
Animals characterized by several jointed legs and a
hard outer skeleton, e.g. spiders, ticks, mites, and
insects (the group that includes mosquitos).
bioassay
Assessment of the efficacy and persistence of an
insecticidal treatment by exposing mosquitos of
known susceptibility to a treated surface or area for a
standard period of time.
chloroform
A chemical used for immobilizing mosquitos to
facilitate their handling. It is toxic and should be used
with extreme care.
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chloroquine
The drug most commonly used for treating malaria.
1
climatic data
Information on weather patterns, such as maximum and
minimum temperatures, rainfall, and relative humidity.
culicine mosquitos
Most mosquitos that are not anophelines fall into this
group. Culicine mosquitos are not vectors of human
malaria, but may transmit other diseases (e.g. dengue
fever, filariasis, Japanese encephalitis).
drop net
A large net on a rigid frame, which is dropped over
vegetation to collect specimens of outdoor-resting
mosquitos.
ecological area
A well defined geographical area, for example a
tropical rain forest, characterized by certain assem
blages of plants and animals (including insects).
eggs
Fertilized female reproductive cells with nutrient ma
terial, deposited by female mosquitos and developing
in water to produce free-swimming larvae.
endophilic
Tending to rest indoors, whether by day or by night.
exit traps
Devices typically placed over doors and windows of
houses or animal shelters to catch mosquitos leaving
these buildings.
exophilic
Tending to rest outdoors, whether by day or by
night.
feeding habits
Habits determining the times and places of feeding
and the sources of blood meals for mosquitos.
gametocyte
These are sexual forms of the malaria parasite that
develop within red blood cells. The male gametocytes
are called microgametocytes, the female macrogameto
cytes.
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73
Entomological field techniques for malaria control. Learner's Guide
gonotrophic stage
The condition of female mosquitos during blood
ingestion and ovarian development.
host preference
The preference of a mosquito for a particular type of
host, human or animal. (To be distinguished from
simple readiness to feed on a given type of host when
no other is available.)
insecticide
A chemical designed to kill insects.
insecticide resistance
The ability of a mosquito to survive contact with an
insecticide in quantities that would normally kill a
mosquito of the same species.
instars
Stages of insect growth and development. In mosqui
tos there are four larval instars, each terminating with
the shedding of the skin.
larva
The second stage in the life cycle of the mosquito.
Larvae develop within the eggs and emerge when the
eggs.hatch. They swim and cat small organisms on
the surface of water.
larval habitat
The type of aquatic environment in which mosquito
larvae are typically found.
larval survey
The process of searching for mosquito larvae in a
defined area. Larvae are collected and taken to the
laboratory for identification.
life cycle
The stages of development through which a plant or
animal passes during its life. For mosquitos these
stages are: egg, larva, pupa, adult.
A parasite that lives in the blood of humans and
animals. Malaria parasites of humans belong to the
group with the scientific name Plasmodium.
malaria prophylaxis
Measures taken for protection against malaria, e.g.
administration of a drug that prevents a person from
becoming infected with the disease.
malaria survey
A study designed to determine the number of people
carrying malaria parasites in their blood.
malaria transmission
The process by which a vector mosquito transfers
malaria parasites from an infected person to one who
is uninfected.
malariologist
A person who has studied and is knowledgeable
about malaria and its control.
man/vector contact
The number of times a person is bitten by a vector
mosquito, normally expressed as the number of bites
per person per night.
mortality rate
The percentage of mosquitos that die within a speci
fied period of time.
mosquito repellent
Any substance producing a negative response in
mosquitos, causing them to avoid a close approach
(such as alighting on the skin of a host animal or
entering a treated room).
74
i
Explanation ol terms
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oocyst
Fertilized female cell (zygote) after the ookinete
penetrates and encysts in the wall of the mosquito
stomach. This cell undergoes division to produce
sporozoites.
ookinete
Motile (mobile) stage of the malaria parasite resulting
from fertilization of the macrogametocyte by microgametocyte(s) in the mosquito gut. After passing
through the gut wall, it becomes the oocyst.
operational surveys
Field surveys conducted periodically to evaluate or
monitor malaria control methods and their effects.
outbreak of malaria
A sudden increase in the number of people sick with
malaria in a particular area (village, town, district).
parasite
A plant or animal that lives on or in, and derives its
nutrients from, a host plant or animal.
parasite density
The number of malaria parasites in a specified volume
of blood.
parous rate
The proportion of female mosquitos that have laid
eggs at least once.
peak biting time
The period during the biting cycle of a given anopheline species when the largest number of females take
blood meals.
precipitin tests
Tests in which blood from the stomach of fed mos
quitos is analysed to identify the host species.
preferred breeding
sites
Sites suitable for egg-laying and satisfactory for all
aquatic stages of development.
seasonal prevalence
The number of cases of infection in relation to the
unit of population in which they occur (a static
measure) at different times of the year.
pupa
The third stage in mosquito development, emerging
when the fourth larval instar sheds its skin. Pupae
swim but do not feed.
random sampling
The sampling process whereby each unit in the
population has an equal chance of being selected.
rate of infection
The percentage of mosquitos carrying malaria para
sites. The relationship between the sporozoite rate
and the total infection rate is closely dependent on
longevity, which may be estimated from these two
factors. If an estimation of longevity is required,
examination for sporozoites may be supplemented by
careful examination for oocysts, and separate rates for
oocyst, sporozoite and total infection may be re
corded.
red blood cells
Cells in the blood that carry oxygen from the lungs
to the tissues and carbon dioxide from the tissues to
the lungs. It is the red blood cells that are attacked by
the malaria parasite.
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75
I
Entomological held techniques for malaria control Learners Guide
relapse
A renewed manifestation of clinical symptoms and/or
parasitaemia associated with malaria infection, sepa
rated from the previous manifestation by an interval
greater than one reflecting the normal periodicity of
paroxysms.
representative sample
A sample that gives an indication of the composition of
the whole population.
resistance to drugs
The ability of a parasite to live in the presence of a drug
that would normally kill members of the same species.
resting habits
The habits determining the places and times of day or
night that mosquitos rest.
salivary glands
The glands that produce the saliva injected when a
mosquito bites, which prevents blood from clotting
while the mosquito feeds.
sampling
It is usually impossible to collect every individual in a
population, such as .all the mosquitos in a village.
Sampling means taking a small, representative pro
portion of the total.
species
A fairly well defined, interbreeding group of plants or
animals.
species infection
rate
The percentage of a species of mosquito carrying
malaria parasites.
sporozoite
The final stage of development of Plasmodium in the
mosquito; this is the infective form of the malaria
parasite, occurring either in a mature oocyst before its
rupture or in the salivary glands of a mosquito.
surveillance
The monitoring of changes in the numbers of malaria
cases over a period of time.
susceptible
The term applied to a vector against which an
insecticide is completely effective.
susceptibility test
A test to assess the extent to which a population of a
given mosquito species is susceptible to a particular
insecticide. Samples of mosquitos are exposed to
known concentrations of an insecticide for specified
periods.
taxonomic study
Identification of species and their genetic relationship
to one another.
transmission
The process whereby the malaria parasite is carried
from person to person by mosquitos.
trap nets
Nets used to sample the living mosquito population.
By permitting access to a bait but restricting move
ment away from it, trap nets tend to concentrate
female mosquitos near the bait.
vector
An insect or other animal that carries a disease from a
plant or animal to another plant or animal of the same
species which is free of the disease. Anopheline
mosquitos are the vectors of human malaria.
76
Exp'anauon of terms
vector density
The number of a given vector species present. It may be
expressed in relative terms (e.g. the biting density in
relation to the human host) or in absolute terms (e.g. the
number present in a room, cattle-shed or artificial
shelter).
zygote
The product of the union of a male and a female
gamete.
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DOCUMENTATION
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WHO publications of related interest
Pnce*
(Sw. fr.)
Basic malaria microscopy
Part I: Learner's guide. 1991 (72 pages)
Part II: Tutor's guide. 1991 (69 pages)
14 —
14-
The biology of malaria parasites
Report of a WHO Scientific Group
WHO Technical Report Series, No. 743. 1987 (229 pages)
32-
WHO Expert Committee on Malaria
Eighteenth report
WHO Technical Report Series, No. 735. 1986 (104 pages)
14-
Malaria control as part of primary health care
Report of a WHO Study Group
WHO Technical Report Series, No. 712. 1984 (73 pages)
8-
Vector control in primary health care
Report of a WHO Scientific Group
WHO Technical Report Series, No. 755. 1987 (61 pages)
9.-
Manual on environmental management for mosquito
control, with special emphasis on malaria vectors
WHO Offset Publication, No. 66. 1982 (283 pages)
22-
Malaria control and national health goals
Report of the Seventh Asian Malaria Conference
WHO Technical Report Series, No. 680. 1982 (68 pages)
6.-
Educational handbook for health personnel, 6th ed.
WHO Offset Publication, No. 35. 1987 (354 pages)
34-
Self-learning materials and modules for health workers.
A guide for their development, utilization and evaluation
SEARO Technical Publications, No. 6. 1985 (18 pages)
3-
Further information on these and other World Health Organization publications
can be obtained from Distribution and Sales, World Health Organization,
1211 Geneva 27, Switzerland.
* Prices in developing countries are 70% of those listed here.
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