Crop protection 4

General Physiology and toxicology

Exercise 5
UPTAKE OF SALTS BY AQUATIC INSECTS
I.

Introduction
Mosquito larvae are found in fresh brakish and saline water. Beadle
in 1939, was first to demonstrate that the larvae found in saline posses
water specific osmoregulatory mechanisms not found in fresh water forms.
Later, it has been shown that these distinct. Mechanisms are results or
morphological and physiological specializations, fresh water larvae
produce dilute urine to rid the body active ion uptake through the cuticle,
larvae from saline water drink external medium to maintain body volume
and produce hyperosmotic urine to eliminate ingested ions.

II.

Objectives:
To demonstrate physiological and morphological adaptations of
mosquito larvae to water with different concentrations.

III.

IV.

Materials:
100ml beakers/bottles
distilled water
aedes aegpyti egg
NaCl
Mosquito
Nylon netting to cover beakers
Rubber bands
Procedure
1. Prepare to following solutions:
a. Distilled water

b. Stock solution of 0.65% NaCl w/ 1 to 6.5 grams of NaCl, add 100ml

w/ distilled water
2. Place the solution in beakers and level accordingly. Make 3 replicates
per treatment
3. Place 10 first instar larvae of aedes aegyti in each of the 4 beakers
4. Feed the larvae but donot over feed
5. Cover the beakers w/ fine fine nylon netting held in place by rubber
band
6. Observe the larvae record the behavioral differences in the larvae in
the 3 beakers. Rate, of swimming, direction of swimming, preffered
location in water
7. When the larvae 4th instar, collect the larvae in 3 beaker-color size,
markings: pay attention to the size of anal papillae of the larvae in 3
beakers measure each papillae of each larvae
8. Repeat the experiment using cullex , pipiens/ faciatus larvae
9. Prepare your report answering the following question
a. Are there behavioral, difference in the larvae reared in the solution
b. How do you account for the difference in size of the anal papillae of
the larvae reared in the 3 solution
c. How do you account for morphological change in the cullex larvae

V.

reared in 3 solutions
d. Relate this finding to natural habitat in w/o species are found
10. Discuss your results include a review of literature and reference

THE ADAPTATION OF MOSQUITO LARVAE

TO SALT WATER
BY V. B. WIGGLESWORTH, M.A., M.D.
(From the Department of Entomology, London School of Hygiene and Tropical
Medicine.)
(Received x 5th April, x932. )
(With Two Text-figures.)
IT is well known that the larvae of certain mosquitoes can thrive both in fresh
water and in water of a high degree of salinity, while the larvae of other
mosquitoes are quickly killed by salt water; and that such differences may exist
even between local races of the same species (Evans, 93 t). It is evident hat we

have here to deal with a physiological adaptation, and it was the object of the
present work to discover what the nature of this adaptation might be. The
physiological properties of larvae of the yellow-fever mosquito (Aedes
(Stegomyia) argenteus Poir), which were accustomed tosalt water, have been
compared with those same properties in normal arvae. It so happens that this
particular larva is unable to tolerate very salt water; but it is probable that the
relatively small degree of resistance which it can acquire is of the same kind as
that occurring in more tolerant species, and therefore the mechanism of this
resistance may throw light upon the general problem.
LETHAL CONCENTRATION OF SALT FOR LARVAE REARED IN FRESH
WATER.
Sodium chloride. Fourth stage larvae (i.e. larvae in their final instar),
reared in fresh water, were transferred to sodium chloride solutions of various
strengths at 24 C. They are not affected by solutions of 0. 9 per cent and less.
In -o per cent solutions many larvae die within about a week (although afew may
survive permanently), and the mortality increases with rising concentration. At .
and .2 per cent the larvae nearly all die in 4-7 days. At .3 per cent. they die
within 72 hours, and at .4 per cent. within 48 hours. Individual variations are
considerable.
Artificial seawater. Experiments were carried out under the same
conditions using diluted seawater in place of sodium chloride. The concentration
of seawater may be conveniently expressed in terms of those solutions of sodium
chloride which have the same osmotic pressure, this being calculated
approximately on the assumption that all the salts are completely ionised. These
equivalent concentrations are shown throughout in inverted commas.
The artificial sea water used had the following composition: NaC1, 2-83 per
cent.; KC1, o'o76 per cent.; MgCle, o-5ox per cent.; CaCI, o. 122 per cent. This
has an osmotic pressure approximately equal to 3'5 per cent. sodium chloride.
No bicarbonate was added to this mixture, which was therefore more acid than
natural sea water.

28 V. B. WIGGLESWORTH
It is found that the larvae are unaffected by a concentration of" . per
cent."; at "x-2 per cent." there is a considerable mortality in the course of a week,
but many of the larvae survive. At "x'3 per cent." very few survive; at "x'4 per
cent." they are nearly all dead in a week; at "x'5 per cent." all die within 4 days,
and at "x-6 per cent." in about 4 8 hours. There is much individual variation (for
example, very rarely a larva may survive transference to "x- 4 per cent." sea
water, and live apparently indefinitely), but it is evident that sea water is
considerably less toxic than pure sodium chloride, x-2 per cent. NaC1 being
about equally toxic with "x'5 per cent." sea water.
EFFECT OF SALTS ON THE GROWTH OF NEWLY HATCHED LARVAE.
One of the difficulties in studying the effect of the composition ofthe water
on the growth of mosquito larvae is the indirect effect which this composition may
have by its influence on the micro-organisms upon which the larvae feed. In the
present work this difficulty has been avoided by feeding the larvae on dried and
finely powdered serum (Loeffler's dried serum) upon which they thrive extremely
well.
An equal amount of this (as measured on the point of a knife) is added to
equal (to c.c.) volumes of the fluid to be tested, and an equal number (about half
a dozen) newly hatched larvae placed in each. Sodium chloride. A series of
concentrations of sodium chloride ranging from distilled water to 3 per cent. was
employed, being kept at 24 C. The larvae grow rapidly in pure water, being all
at the fourth or final instar in 5 days. Growth is very slightly delayed below 0-8
per cent. NaC1. There is greater inhibition at 0- 9 per cent., larvae being at the
second or third instar in 5 days. At x-o per cent. there is often considerable
mortality and the growth of the survivors is extremely slow, there being almost no
growth, and larvae still in the first instar at the end of 5 days. At x-x per cent. the

larvae fail to grow, and die in about 4 8 hours. These are all dead in about 2
hours in '5 per cent., and in about t hour at 2-o per cent. and over.
The same results are obtained if the eggs are allowed to hatch in these solutions.
Thus they hatch normally in x-2 per cent. NaC1; but the larvae all die within 2
days. Artificial sea water. Larvae reared under the same conditions in artificial
seawater at various dilutions develop at about the normal rate up to a
concentration of "x.x per cent" (in terms of the concentration of NaCI having the
same osmotic pressure). At" x-2 per cent." growth is slightly inhibited. At "x'3 per
cent." Many of the larvae die in a few days and the growth of the survivors is very
slow. At "x-4 per cent." all die in about 4 8 hours; and at "x' 5 per cent." most are
dead within 2 4 hours.
ADAPTATION OF LARVAE TO SALT WATER.
Sodium chloride. Newly hatched larvae were placed in a weighed amount
of 0. 9 per cent. NaC1 in an open vessel with a supply of dried and powdered
serum for food, and the concentration of salt allowed to increase slowly by
evaporation, the mixture being stirred each day. The specific gravity of the initial
fluid being known, the concentration at any stage can be calculated from the
weight. Adaptation of Mosquito Larvae to Salt Water 29. The larvae do not
become very resistant to sodium chloride; and if this is allowed to reach a
concentration f about I'3 per cent. during xo days, most of the larvae are dead or
moribund. But if the evaporation is arrested when the concentration has reached
-o or -x per cent., or if the larvae are kept in .o per cent. NaC1 from the outset,
it can always be noted that for some days they show practically no growth; but
when growth as once begun it proceeds fairly rapidly. This suggests that some
sort of adaptation is occurring, and it will be shown later that such is the case.
,Sea water. Iore striking adaptation can be obtained with artificial sea water.
The experiments have been carried out as with sodium chloride, the larvae being
started off in sea water diluted three times. Under these conditions, some of the

larvae will survive until the water has reached a concentration of about "2-2 per
cent." in 3 weeks. If kept at this concentration, however, they die in a few days.
On the other hand, if the evaporation is arrested when the concentration is
about "'9 per cent." seawater, the larvae will survive for at least a week. These
larvae grow extremely slowly and it is doubtful if they would ever reach maturity;
but there is no doubt hat they have become adapted to the sea water, for we
have seen that larvae reared in fresh water die within 4 8 hours in sea water of
this concentration. In sea water equivalent to "'5 per cent." NaC1 the adapted
larvae have remained alive for 3 or 4 weeks.
CHARACTERS OF LARVAE ACCUSTOMED TO SALT WATER.
General appearance. Larvae accustomed to z per cent. NaC1 show no
obvious difference from larvae in fresh water. On the other hand, larvae in sea
water equivalent to "'75 per cent." NaC1 have the body considerably shrunken,
so that the indentations between the abdominal segments are more pronounced
than usual, and the head sinks into the thorax. This shrunken appearance
becomes more marked the greater the concentration of the salt. Intestine. The
gut often contains less solid matter and more fluid than in fresh water, both inside
and outside the per trophic membrane. The cells of the caeca and the mid-gut
appear normal. atrial gills. The anal gills may sometimes be slightly deformed;
but usually they are if the normal size and shape. The cells may be perfectly
normal or just a little thickened (Fig. z A and A'). More often, however, they have
the appearance shown in Fig. z B and B', where the cells are greatly thickened
so that they extend far into the lumen of the gill. The cytoplasm, which contains
large vacuoles, is separated from the cuticle but bound to this by conspicuous
strands. Sometimes one or more of the gills are normal and the others have the
cells swollen in this manner. (The normal structure and properties of the anal gills
have already been described (Wigglesworth, 932 a).) Malpighian tubes. The
cells of the Malpighian tubes are usually less granular than normal, both in larvae
reared in z per cent. NaC1 and in diluted sea water. Sometimes they contain in

the lumen a considerable amount of solid matter, Adaptation of Mosquito Larvae

to Salt Water 29 The larvae do not become very resistant to sodium chloride; and
if this is allowed to reach a concentration of about '3 per cent. during xo days,
most of the larvae are dead or moribund. But if the evaporation is arrested when
the concentration has reached -o or x.x per cent., or if the larvae are kept in x'o
per cent. NaCI from the outset, it can always be noted that for some days they
show practically no growth; but when growth has once begun it proceeds fairly
rapidly. This suggests that some sort of adaptation is occurring, and it will be
shown later that such is the case. Sea water. More striking adaptation can be
obtained with artificial sea water. The experiments have been carried out as with
sodium chloride, the larvae being started off in sea water diluted three times.
Under these conditions, some of the larvae will survive until the water has
reached a concentration of about "2-2 per cent." in 3 weeks. If kept at this
concentration, however, they die in a few days. On the other hand, if the
evaporation is arrested when the concentration is about "'9 per cent." sea water,
the larvae will survive for at least a week. These larvae grow extremely slowly
and it is doubtful if they would ever reach maturity; but there is no doubt that they
have become adapted to the sea water, for we have seen that larvae reared in
fresh water die within 4 8 hours in sea water of this concentration. In sea water
equivalent to "'5 per cent." NaC1 the adapted larvae have remained alive for 3 or
4 weeks.
CHARACTERS OF LARVAE ACCUSTOMED TO SALT WATER.
General appearance. Larvae accustomed to per cent. NaC1 show no
obvious difference from larvae in fresh water. On the other hand, larvae in sea
water equivalent to "'75 per cent." NaCI have the body considerably shrunken,
so that the indentations between the abdominal segments are more pronounced
than usual, and the head sinks into the thorax. This shrunken appearance
becomes more marked the greater the concentration of the salt Intestine. The gut
often contains less solid matter and more fluid than in fresh water, both inside

and outside the per itrophic membrane. The cells of the caeca and the mid-gut
appear normal. Anal gills. The anal gills may sometimes be slightly deformed; but
usually they are of the normal size and shape. The cells may be perfectly normal
or just a little thickened (Fig. I A and A'). More often, however, they have the
appearance shown in Fig. B and B', where the cells are greatly thickened so
that they extend far into the lumen of the gill. The cytoplasm, which contains
large vacuoles, is separated from the cuticle but bound to this by conspicuous
strands. Sometimes one or more of the gills are normal and the others have the
cells swollen in this manner. (The normal structure and properties of the anal gills
have already been described (Wigglesworth, 932 a).) Malpighian tubes. The
cells of the Malpighian tubes are usually less granular than normal, both in larvae
reared in x per cent. NaC1 and in diluted seawater. Sometimes they contain in_
the lumen a considerable amount of solid matter,
PHYSIOLOGICAL PROPERTIES OF LARVAE ACCUSTOMED TO SALT
WATER AS COMPARED WITH LARVAE IN FRESH WATER.
It is evident hat the properties of the anal gills must be considerably
changed in larvae accustomed to salt water. For, as already described
(Wigglesworth, 932a), if larvae from fresh water are immersed in per cent.
NaC1 or "-4 per cent." sea water, the cells of the gills swell up enormously. This
swelling is resisted by the inherent elasticity of the cells, due at least in part to the
vertical filaments which tie the cells to the cuticle. In larvae from salt water these
filaments have become conspicuous strands. This suggests that the essential
change in the gills of larvae accustomed to salt water is the increased strength of
these elastic structures.

Crop Protection 4
General Physiology and Toxicolgy

Exercise 6
Growth and Development
Introduction
The life cycle of an organism consists of periods of growth and replication
and during which a single cell, the egg, becomes an adults w/ millions of cells.
These changes w/ are collectively known as development are endocrine control.
The three components of developments namely growth, differentiation and
morphogenesis will invoke hormonal actions given times and place during
ontogeny
The visible expression of growth required insect to molt morphogenetic
changes occupying each steps in the molting sequence may lead to adult form
through a series of larvae stages. The metamorphosis w/c involves external and
molt or at the larval pupal and pupal adult molt
Objectives:
This exercise aims to determine the relationship between the hormones
produced by endocrine glands and the insect population
Materials
1. Housefly larvae (Musa domestica)
a. Young larvae 35 day
b. Old larvae
2. Heavy thread
3. Petri dishes
4. Filter paper/ tissue paper
5. Dissecting dishes
6. Stereomicroscope
7. 1% methylene blue solution
8. Masking tape
9. Marking pen
10. Dissecting
Methods
A. Orientation
1. Immobilize housefly larvae by chilling

2. With ventral side up carefully dissect the larvae so as to expose the

nervous system. Use 2 to 3 drops of 1% methylene blue locate the nerve
tissue, if necessary
3. Draw the location of the right gland and brain in relation to the body
segments
B. Experimental plan
1. Select 400 young larvae and 400 old divide into 25 individual each
2. Ligate each one w/ fine thread at (a) point anterior to the ring gland (b)
point anterior to the ring gland. Keep the 4 replicates for each group
3. Place the larvae in covered petri dishes lined w/ filter paper slightl
moistened w/ water. Do not let the larvae escape. Seal w/ masking tape.
Label the dishes accordingly
4. Observe the result (Region that pupated) twice each day for 3 days
5. Record your data in tabular information
6. Discuss your result review of Related literature and reference

The mosquito has four distinct stages in its life cycle: egg, larva, pupa, and
adult. The adult is an active flying insect, while the larvae and pupae are aquatic
and occur only in water. Depending on the species eggs are laid either on the
surface of water or are deposited on moist soil or other objects that will often be
flooded. One method of classifying mosquitoes, which is important in the control
of the larval stage, is by the type of habitat in which the eggs are laid. Those
species that lay eggs singly on the moist soil usually near the edge of temporary
pools of water are known as flood water mosquitoes. These eggs only hatch after
they

have

been

flooded

by

water. Psorophora, Aedes,

and Ochlerotatusmosquitoes are floodwater mosquitoes which are most

abundant shortly after spring rainfall. Those species that lay eggs on the surface
of the water, either clumped in rafts or as single floating eggs, are known as
permanent water mosquitoes. Anopheles, Culiseta, and Culex are permanent
water mosquitoes found in Oklahoma. Floodwater mosquitoes are usually pests
in April and May in Oklahoma, with permanent water mosquitoes being problems
later in the summer.

Literature Review
Price (1963, 1975) studied the growth in the number of scientists, scientific
journals, and papers over the past two centuries, finding that the numbers
doubled every 15 years. Since then, literature growth studies have become very
common in the field of bibliometrics and infometrics. Studying growth patterns in
the NLM's serials collection and inIndex Medicus journals between 1966 and
1985, Humphreys and McCutcheon (1994) concluded that the data appear to
support Price's analysis, which was further developed by Goffman (1966, 1971)
describing it as an initial period of exponential growth, followed by saturation and
slowdown to a steady rate of increase. A similar conclusion was reached earlier
by Orr and Leeds (1964) concerning the biomedical literature. The " Law of
Exponential Growth " has been further dealt with by Tague and others (1981),
Ravichandra Rao and Meera (1992), Egghe and Ravichandra Rao (1992),
and many others. The exponential growth of the literature is described
mathematically by the exponential function YT =a.ebt where YT represents the
size at time t, a is the initial size, and b is the continuous growth rate which is
related to the annual percentage growth rate r, as: r =100(eb-1). Egghe and
Ravichandra Rao (1992) claim, however, that the power model (with exponent
>1) is the best growth model for sciences and technology fields, while the
Gompertz S-shaped distribution fits better databases of the social sciences and
the humanities.
Findings
A total of 25,482 publications represent the literary output in different
formats, in different subjects, and from various nations. However, for more
granular results, the findings have been divided into broader headings.
Source Type
The total database of 25,482 publications represents different formats. A majority
of the publications are journals and journal articles, with a total of 23,830

(93.51%), followed by conference proceedings and trade journals at 755 (2.96%)

and 699 (2.74%), and book series, with a total of 198

The life cycle of a house fly begins in the egg stage. A female house fly is
capable of laying up to 150 eggs in a batch. Over a period of a few days, she will
produce five or six batches of eggs. Female house flies favor damp, dark

surfaces such as compost, manure and other decomposing organic material for
egg laying. House fly eggs resemble individual grains of rice.
Within a day, house fly eggs hatch into larvae, also known as maggots. Maggots
are legless, white insects that feed from the egg-laying site for three to five days.
During this time, maggots molt several times. They then choose a dark place to
pupate.
Fly pupae are similar in function to butterfly cocoons: their hard, brown shells
protect the inactive, developing flies. Over the course of three to six days, the
pupae develop legs and wings, ultimately emerging as full-grown house flies.
Within two to three days, female house flies are capable of reproduction.