1
STUDIES OF REPRODUCTION AND STERILITY
IN DYSDERCUS FASCIATUS Signoret.
by
ARMANDO ANTUNES DE ALMEIDA, Eng. agr. (Lisboa), DIC
A thesis submitted for the Degree of Doctor of Philosophy
in the Faculty of Science, London University.
Imperial College of Science and Technology, Field Station, Ashurst Lodge,
Ascot, Berkshire. January , 1974 2
ABSTRACT
The reproductive organs and mating behaviour in Dysdercus fasciatus
Signoret are described; the existence of a structure considered to be
an erection fluid pump is reported for the first time.
The sterilizing effects of an alkylating agent (tepa) and three
s-triazines on adult males of D. fasciatus have been investigated; also
the effect of tepa upon nymphal stages.
Dysdercus males were completely sterilized when topically treated 2 with 5 µg--of tepa or exposed to a residual deposit of 15 peg/cm . Fertility
was recovered subsequently at rates which were dose-dependent.
Higher doses of tepa were toxic to third and early fifth year instar
nymphs; the LD for the latter was ng. The reproductive potential 50 53 of surviving adults was effected. Late fifth instar nymphs were much
less susceptible, being unaffected by doses toxic to younger nymphs of the
same instar.
Amongst s-triazines evaluated, tri- and pentamethylmelamine were
effective as chemosterilants; SD values were 24 and 73 ng per male. 95 Similar doses of diamino (2-furyl)-s-triazine did not induce any
sterility.
To study the effect of dietary gossypol on reproduction, cotton
'stainer bugs were reared on four different strains of cotton seed,
varying in their content of gossypol, a polyphenolic compound known to
be toxic to, some non-ruminant animals and to some lepidopterous larvae.
The numbers of mating-days and batches of eggs were reduced when
D. fasciatus was reared and maintained on cotton seeds with a very
low or high content of gossypol. The number of resulting adults,
sex ratio (male/female), fecundity, and fertility were reduced
significantly when cotton stainer bugs were reared and maintained on 3
the latter cotton seeds, suggesting that abnormal contents of gossypol are harmful to this species, whether very low or very high.
D. fasciatus adults reared on M-8 glandless and 247-1 high gossypol weighed significantly less than those on Ashurst Lodge cotton seeds having an intermediate concentration of gossypol.
When small doses of gossypol were applied topically to young adult male Dysdercus which were subsequently mated with normal females, fertility was increased in males fed on low gossypol seeds, decreased in males fed on "normal" gossypol seeds, and unchanged in males fed on high gossypol seeds, where fertility was already low.
The quantities of gossypol ingested by Dysdercus when feeding on
Ashurst Lodge cotton seed, and the quantities in the gut and in the frass were determined by chemical analyses. It was found that at 27°C gossypol -casses through the gut in 2.6 days, and approximately 45% of ingested gossypol passes out in the frass.
The mean daily intake of food by adult males was determined and feeding preferences were investigated. Normal glanded seed was preferred to glandless seed but the bugs showed no preference between high gossypol and glandless seed. The quantity of food consumed was significantly smaller when high gossypol and glandless seeds were offered.
The significance of these results and their possible implications for cultural control measures were discussed. CONTENTS
ABSTRACT •• • • 2
GENERAL INTRODUCTION •• • • 8 INSECT MATERIAL AND BREEDING METHODS •••• 12
PART ONE THE CHEMOSTERILIZATION OF THE COTTON STAINER BUG.... 15
DYSDERCUS FASCIATUS Signoret
SECTION A.Structure of the reproductive system • • • 16
Introduction • • • • 16
Material and methods •••• 17
Male reproductive system •• • • 18
Female reproductive system .••• 24
Mating behaviour •••• 28
SECTION B.The effect of some potential chemosterilants •••• 30
INTRODUCTION •••• 30
Alkylating agents. Antimetabolites. Phosphoramides
and s-triazines. Miscellaneous compounds. Insect
hormones and analogues.
MATERIAL AND METHODS .... 43
1. Experimental methods .... 45 1.1. Method of immobilization .... 43
1.2. Method of topical application .... 43
1.3. Residual film technique .... 43
1.11. Post-treatment procedure .... 44
2. Estimation of the median sterilizing and lethal doses .00* 45 2.1. Sterilizing doses .... 45
2.2. Lethal doses 46 3. Statistical methods
4. Chemosterilants and solvents
RESULTS
1. Tepa treatments on 3rd instar nymphs, by topical application .... 48 2. Tepa treatments on 5th instar nymphs, by topical
application .... 51
3. The effect of tepa on adult males, applied
topically .... 55
4. The effect of tepa on the nymphal stage in the
F generation .... 60 1 5. Residual action of tepa on male of Dalercus .... 60 6. Effect of s-triazines, when applied topically,
to .Males of D. fasciatus .... 65
DISCUSSION • • • • 73 Male sterility. Recovery of fertility. Other effects.
Effects of tepa treatment on nymphs.
PART TWO THE EFFECT OF GOSSYPOL ON DYSDERCUS FASCIATUS Signoret. .... 79
SECTION A.Influence of the _glandless and glanded cottonseeds
on development, fecundity and fertility fIll f0 8o INTRODUCTION AND REVIEW OF LITERATURE .... 8o Occurrence of gossypol. The effects of dietary gossypo]
on animals. The effects of gossypol on insects and
mites. Determination of free gossypol. MATERIAL AND METHODS .... 88 1. Cottonseeds .... 88 2. Gossypol .... 88 3. Determination of free gossypol in cottonseeds Reagents and apparatus. Calibration curve.
Preparation of the samples. Gossypol
determination. .... 88
. Experimental methods .... 92
5. Statistical methods .... 93
RESULTS .... 94
1. Gossypol content of the cottonseeds .... 94
2. Nymphal development, fecundity and fertility of D. fasciatus reared and maintained on glandless and glanded cottonseeds .... 95 3. Reproduction rate of adults fed on different
cottonseeds, in relation to their
content of free gossypol, ....102
4. Comparative index ....102
DISCUSSION ....106
' SECTION B. The effect of gossypol applied topical12211
nymphs and adults.
INTRODUCTION
MATERIAL AND METHODS
1. Topical application of gossypol on nymphs and adults ....112
2. Evaluation of toxicity ....112
RESULTS ....114
1. Topical application on 5th instar nymphs
2. Topical application on males
3. Mortality effects
DISCUSSION
SECTION C. Uptake and excretion of gossypol 0 0 0 0127
INTRODUCTION 4,00.12 7
MATERIAL AND METHODS •• ••128 1. Conversion factor. ....128
2. Amount of food eaten. ....128
3. Comparative preference, ....129 4. Free gossypol in the frass.
5. Free gossypol in the gut. ....131 6. Statistical methods ....131
RESULTS ....132
1. Amount of food consumed ....132
2. Free gossypol in the frass,
3. Free gossypol in the gut ....144
4. The comparative preference of D. fasciatus for glandless and glanded cottonseeds....144
DISCUSSION ....149 The comparative preference. Gossypol in the frass.
GENERAL DISCUSSION ••••151
Breeding methods. Fecundity, fertility and gossypol.
Topical application of gossypol. Dysdercus control
SUMMARv ....161
ACKNOWLEDGEVENTS ....168
REFERENCES ....170 GENERAL INTRODUCTION
The control of insect pests by the direct application of
insecticides is the most general method of plant protection.
The effectiveness of control is normally assessed by the mortality
rate of the pest species without regard to the desiquilibrium
provoked in the ecosystem particularly in relation to parasites
and predators.
The extensive use of insecticides has resulted in problems
concerned with localized residues, which form a potential health
hazard, with more widespread pollution, which may cause harm to fish and wildlife, and with the development of strains resistant
to insecticides. An alternative method of pest control, suggested by Knipling in 1937 (Lindquist, 1955), was the possibility
to use radiation to sterilize insects. Knipling's ideas were only
made practicable in the early fifties by the discovery that insects could be sterilized with X- and w-radiation. The theoretical advantages of the release of sterile insects into the natural
populations compared with insecticide treatments for pest'control were reviewed by Knipling (1955, 1959, 1960, 1962 and 1963). The application of this method to eradicate the screw-worm fly from the island of Curacao by the release of sterilized adults was a success and prompted an examination of this method of control for other pests (Baumhover et al., 1955; Knipling, 1960). The use of this method is however limited to a very few species by a number of biological and environmental requirements. For example it would be necessary to rear insects of the pest species on a very large scairs, and agricultural pests are crenerallv difficult to rear in sufficient number for release while their environment frequently
extends over vast areas. The procedure adopted must induce
ActWV 5O-„IA' 14.44,14 .4as )ibtoor.v."4.4. Ao.:tws 25A 11.A.3.1(■\11 doWVI • complete sterility, without otherwise enfeebling the male, while
the release technique must allow even distribution of the sterile
insects with the active population. The sterile male technique
requires the mass production and release of millions of sterile
insects, which may be costly and in many instances not very
practicable.
Because of these difficulties, the use of radiation to control
agricultural pests has been very limited, but chemical sterilization
of insects should be a better alternative.
At the end of fifties and beginning of sixties it was found
possible to sterilize insects by the use of certain chemical
products that affected partially or totally the reproductive
capacities of insects to which they were applied, at doses which
did not produce apparent toxic side-effecAs, as an alternative
to irradiation. These chemicals have been named chemosterilants.
They can be used to sterilize mass-reared insects for subsequent
release or conceivably they can directly sterilize a proportion
of the native population by being incorporated into baits or used
in conjunction with insect attractants. This latter method could
therefore eliminate the inconvenience of mass rearing, sterilization
and subsequent release of large number of sterile insects in the
environment (Smith et al., 1964; La Brecque and Smith, 1968;
Proverbs, 1969).
It should be emphasized however that control by the use of
chemosterilants is still very much in the experimental stage.
With the exception of insect hormone analogues, those at present
available have proved to be too toxic to humans for use in the e field. 10
Part one of this thesis seeks to provide information on the
possibilities of sterilizing the cotton stainer bug Dysdercus
fasciatus Signoret (Hemiptera: Pyrrhocoridae) by using chemosterilants.
This species is a very important cotton pest; direct damage is
caused to both developing and ripe seeds, and indirectly by the
introduction of pathogenic organisms into the unopened bolls
which results in staining the lint, thus affecting the quantity
and the quality of the yields. The aim was to investigate the
effects of chemosterilants applied to adult males in relation to
the fecundity and fertility of the untreated females to which
they were subsequently mated. The effect of tepa on 3rd and
5th instar nymphs of this species was also studied to find out
whether nymphal development would be affected and whether the
surviving adplts-would be capable by reproduction.
During the course of these experiments it was observed that
natural sterility in untreated insects often occurred. Since
this may have been the result of inbreeding a new strain was
acquired from the Makoka. Research Station in Malawi. The
possibility that dietary factors ought also to be incorporated
led to a consideration of the effects of gossypol, a terpene
. phenol harmful to some species of insects, which is present in
varying amounts in cottonseeds on which the insect feeds.
The second part of this work is therefore concerned with
the study of the effect of gossypol, either incorporated in the
diet or applied topically, on the reproductive ability of
D. fasciatus, and is presented in three sections. In Section A
the amount of free gossypol in cottonseeds was determined and
its influence on development, fecundity and fertility of he
nymph and adult cotton stainer bugs was investigated. The 11 effectiveness of gossypol, when applied topically to nymphs and males, and also its mortality effect was studied in Section B.
In Section C the amount of food ingested and the comparative
preferences of bugs for glandless and glanded cottonseeds (1) was studied, the uptake and excretion of free gossypol was examined and the amount of free gossypol in the gut of the male was determined.
In all the experiments described in Part two, natural sterility was very low or almost unnoticeable, and therefore inbreeding was probably the factor responsible for the high natural sterility that was initially found in the first cultured strain. The same phenomenon was reported by MacGill (1942) for Dysdercus howardi Ballou.
(1) Glanded oottonseeds have pigment glands which contain gossypol scattered throughout the tissue of the embryo.
Glandless cottonseeds have no pigment glands. 12
INSECT MATERIAL AND BREEDING METHODS
The experiments were carried out with nymphs and adults of
Dysdercus fasciatus Signoret. The insect was first obtained in culture from the Silwood Park insectary, where it had been reared for at least five years, and from this a stock was built up at
Ashurst Lodge.
Hodjat (1963) reported that fecundity decreased with shortage of water. To prevent this occurrence optimal humidity conditions were provided; a relative humidity of 70 + 5% was maintained in the constant temperature room, in which the culture containers were kept as was recommended by Hargreaves and Taylor (1938), cited by A. M. Brunt (1970). This gave the quickest rate of development and the greatest total number of both eggs and egg batches. The cultures were incubated at 27 + 0.5°C.
The insects were Cultured in two perspex aquarium tanks,
40 x 30 x 35 cm., with a layer of peat (3 - 4 cm. high) at the bottom, which had been soaked in boiling water and squeezed prior to use, to provide an oviposition site. It was necessary to spread it loosely to facilitate the digging and oviposition by the female; 15 to 20 pairs of adults were placed in each container.
Cottonseed, soaked overnight, was provided as food, and fresh water was made available in the form of soaked cotton wool. Three
times each week the food was changed to prevent the development of
diseases in the culture and the peat was sprayed with water daily
to help to maintain a humidity favourable for the development
of eggs and nymphs; the 1st and 2nd instars being particularly
sensitive. Dead insects were removed and destroyed in each
container daily and a plastic net was provided as support to
facilitate moulting. 13
It was noticed that many clutches of eggs were attacked by fungi and failed to hatch. This could possibly be attributed to
the existence of fungi growing in the peat. For this reason, sterilized peat was used, but without good results since the eggs
continued to be attacked by fungi, presumably from spores existing in the air of the constant temperature room. Subsequently, the chaat peat was sprayed with copper oulphotc (Perenox) at a concentration
of 1.5 g/1 in water, before initiating the culture (personal
advice from Dr. R. J. Wheeler, Botany Dept., Imperial College).
Later, it was observed that healthy eggs were not attacked by
fungi and they hatched normally. The appearance of the fungi in the eggs was therefore regarded not as cause for the failure
of the egg hatch but as a consequence of the eggs being sterile,
in which state they provided good conditions for fungi to grow. ctuJukadv. 4 Under the conditions described theXlife cyolc of D. fasciatus
was 55 - 69 days for the females and 82 - 113 for the males. The
duration of each stage can be seen in Table 1.
3) mailer., Table 1.X Life-evei-e of Dysdercus fasciatus Signoret at 27 .4- 0.50C and 70 5% relative humidity.
Duration (days)
Male Female
Egg 6 - 7 6 - 7
1st instar 2 - 3 2 - 3
2nd instar 3 - 5 3 - 5
3rd instar 4 - 6 4 - 6
4th instar 5 - 6 5 - 6
5th instar 7 - 8 9 - 11
Adult 55-78 28 - 31
TOTAL 82 - 113 55 - 69 During the course of the chemosterilant experiments it was
noticed that natural sterility was very high. In an attempt to
eliminate this inconvenience it was decided to replace the culture
with fresh stock imported as adults from Malawi. Since in-
breeding was probably the main reason for such high natural
sterility, attempts were made to minimize this effect in the new
culture by rearing the insects in a different way. Twelve pairs
of previously mated adults were selected for egg laying and each
of them was placed in a plastic sandwich box (19 x 11 x 8 cm.).
From each pair, the second batch of eggs was chosen and after
incubationi approximately one third of the nymphs were transferred
to the perspex aquarium tank,providing a total of 300 - 400 nymphs
per tank derived from 12 pairs of adults.
The bottom of each plastic sandwich box was covered with
filter paper, into which were placed two small plastic containers;
one for soaked cotton wool and the other with moist peat to provide
an oviposition site for the female on top of which were placed
cottonseeds as food.
This method was an improvement on the original culture
procedure, with the added advantage that as the eggs were all from
• the same day, the adults obtained were more or less of the same
age and subsequently the population used in the experiments was
more homogeneous in this respect. 15
"It would be most unfortunate to relegate the success of the sterilization method to the dormant category of "spectacular", or to treat it as one of those exceedingly clever tricks of a great magician and to close the lid over it with a final comment: what will they think next!"
A. B. BORKOVEC
Proc. 3rd British Insecticide and Fungicide
Conf., 1965 : 3 - 14.
PART ONE
THE CHEMOSTERILIZATION OF THE COTTON STAINER
BUG DYSDERCUS FASCIATUS Signoret. 16
SECTION A. STRUCTURE OF THE REPRODUCTIVE SYSTEM
INTRODUCTION
The reproductive organs and mating behaviour of Dysdercus fasciatus have not been studied in detail, although Valentine (1953) described the reproductive organs in outline. Therefore, it was considered necessary to study in detail the reproductive system and mating behaviour of this cotton bug before determining the effect of chemosterilants on reproductive ability of this species.
Studies of the structure of the reproductive organs in the
Heteroptera have been presented by several authors and the most important accounts are mentioned below.
An early study of the male genitalia' in Rynchota was presented by Singh-Pruthi (1925), describing the morphology of these organs in both Heteroptera and Homoptera.
Bonhag and Wick (1953) studied the functional anatomy of the male and female reproductive systems of the milkweed bug
Cncopeltus fasciatus.
Dupuis (1955) presented a critical review of the literature on morphology of the genitalia of both sexes of Heteroptera with a glossary and synonymy of the terms adopted by different authors in an effort to bring uniformity to the nomenclature used.
Dupuis and Carvalho (1956) have given a general account of the structure of the female genitalia in the Heteroptera.
Pendergrast (1957) presented another study of the reproductive organs of the Heteroptera, which can he considered a preliminary investigation particularly concerned with tne value of these organs as auxiliary characters for the classification of 17
Heteroptera. It was noted that in females the more important
organ in that respect was the spermatheca which showed considerable
diversity.
Scudder (1959) studied the female genitalia of Heteroptera,
and its value in the systematics of the group was again considered.
McDonald (1966) described the male genitalia of 85 and the
female genitalia of 80 species of North American pentatomid bugs,
with the aims of showing more clearly the interrelationships of
the North American genera of the Pentatomoidea and of providing
a basis for a taxonomic review of this group.
Singh.-Pruthi (1925) divided the higher Heteroptera into
three groups on the basis of the differentiation of the endosoma;
here included the Pyrrhocoridae in the "Pentatomid type", where
neither secondary processus nor an internal skeleton are found
in the vesica. This division is still accepted.
During the present study of the. male genitalia of Dysdercus
fasciatus the presence of a membranous structure situated at the
junction of the ductus ejaculatorius with the ductus seminis,
with a pair of dilator muscles attached, was noted. SuCh a structure was not mentioned by any of the authors cited above,
with the exception of Bonhag and Wick (1953) who observed a
similar structure in Oncopeltus fasciatus (Heteroptera : Lygaeidae),
and termed it the erection fluid pump.
MATERIAL AND METHODS
Cotton stainer bugs were reared as previously described.
Mating pairs were suddenly submerged in acetone previously
maintained in the deep-freeze for some time. The insects froze
instantly without disengaging; they were removed and placed in
Bouin's fluid for fixation and preservation. 3.8
The general description of the reproductive organs were
based primarily on live material, but to facilitate the study of
the cuticular parts the specimens preserved in Bouin's fluid were
boiled in 10% KOH to remove non-cuticular material.
Insects for dissection were chilled in the deep-freeze for
6 - 8 minutes and then transferred to dissecting dishes, with
paraffin bases and covered with Ringers' solution.
The morphological terms proposed by Dupuis (1955) for the
description of the male genitalia and the terms used by Dupuis
and Carvalho (1956) for the description of the female genitalia
are adopted here.
MALE REPRODUCTIVE SYSTEM
The male reproductive organs consist of a pair of testes,
vas deferens, a pair of seminal vesicles, one pair of accessory
glands, and an ejaculatory duct (Fig. 1
There are two testes in the cotton stainer bug lying ventro-
laterally in the fifth abdominal segment and they are held in
position by fat body and trachea. Each testis is composed of
seven testicular tubes and are elongate ovoid; they are enclosed
in a membranous sheath pigmented a bright orange red.
The vasa deferentia are the paired ducts that carry the semen
from the testes to the ejaculatory duct; the apical part of each
vas deferens is expanded to form the seminal vesicle and posteriorly
tapers to form a narrow tube. The tubular part of the vas deferens
is partly covered by the mesadenes.
The mesadenia or accessory glands are a pair of mesodermal
glands enveloping the posterior parts of the vasa deferentia. 19 Fig. 1. Reproductive organs of Dvsdercus fasciatus (male).
A - Male reproductive organs,x30.
B Scaning electron micrograph of the bygophore, x177.
b bulbus ejaculatorius.
mg - mesadene alcessory gland.
p paramere.
pc - processus conjunctivae.
v - vas deferens.
ve vesica.
yerzjculn
t - testis. co
Q
0 CV 21 Fig. 1 (continued).
C m Paramero.
I) - Erection fluid pump (efp).
E - Vesica.
bp.br - basal plate bridge.
du.ej ductus ejaculatorius.
du.sm - ductus seminio.
Fg - primary gonopore.
Sg - secondary gonopore. 22
C
0.2 m m
du. e j
0.1 ni rn 23
The ductus ejaculatorius is a median tube that carries the semen from the paired vase deferentia to the primary gonopore, but the anterior hooked end is dilated and the posterior extremity is modified into a pump, the erection fluid pump.
Genitalia. The pygophore is a bulbous structure that terminates the abdomen of the male, corresponding to the ninth segment. It has a dorsal conical process bearing two small black teeth before the apex, and includes the anal tube, the intromittent organ or phallus and the movable copulatory hooks, the parameres, located at each side of the phallus. The phallus lies on the floor of the pygophore and it is inserted by the articulatory apparatus.
Between the main pieces of the articulatory apparatus (the basal
plates), the ductus ejaculatorius enters to the phallus through a foramen in the diaphragm (basal foramen) to open into the ductus seminis. In this junction there is, too, a transparent w (ALL an 4.110.c.i.wwvA, bladderAin the general body cavity, called an erection fluid pump, not previously described in D. fasciatus.
The phallus appears as a tube surrounding the ductus seminis and extending from the basal foramen to the secondary gonopore, consisting of a basal region, the phallotheca, where the two distal segments are invaginated, a median region, the conjunctiva, and a distal sclerotized part, the vesica.
The :narameres have a double-hooked head with the hooks
projecting backwards, over the rim of the ninth segment, and they work in special cavities of the pygophore.
The two sclerotized pieces besides the vesica are the procesmis conjunctivae; probably, they prevent the vesica from twisting laterally, durj.Ing 24
FEMALE REPRODUCTIVE SYSTEM
There are two ovaries of the acrotrophic type each one
consisting of seven ovarioles opening into the lateral oviduct.
These are broad tubes that carry the oocytes from the ovaries
to the median oviduct that is continuous posteriorly with a
wider vagina. Associated with the vagina are the spermatheca
and a pair of yellowish coloured acessory glands (Fig. 2).
The spermatheca, or sperm storage organ of the female,
consists of a convoluted spermathecal duct leading to an
apical sperm receptacle, in the shape of a kidney. Between the
receptacle and the main part of the duct there is a spermathecal
pump consisting of a cylinder of muscle fibres surrounding the
central duct.
Genitalia. The external female genitalia of the cotton bug
arise from the ventral parts of the 8th and 9th abdominal segments
and is of the plate-shaped type (Dupuis, 1955; Dupuis and Carvalho,
1956; Scudder, 1959), where the laterotergites and the gonocoxites
are the more important pieces assuming the function of.a protective
cover to the genital aperture.
. Each laterotergite articulates a gonocoxite or valvifer.
Each gonocoxite bears a gonapophysis or valvulae (blades), the
base of each valvula being differentiated into a slender shank
.called the fibula, which serves as an articulary link.
The four membranous valvulae (gonapophysis) are basally
fused with each other and when they are lowered, during copulation
or oviposition, they enclose a space, the intervalvulae space,
which leads directly into the posterior end of the female
genital chamber. 2 5
Fig. 2. Reproductive organs of Dysdercus fasciatus (female).
A - Ovaries, x 22 .
CO - Common oviduct.
OV - Ovariole. 26
Fig 2 (continued).
B Sperm3thec:1, x58.
C Genitalia, plate-shaped type.
a.tu - anal tube.
b - spermathecal bulb.
d - spermathecal duct.
gx gonocoxite.
It laterotergite.
po.fi - posterior fibula.
t tergite.
v vamina. 27
It 8
It 9
gx8
0.4 mm
28
MATING BEHAVIOUR
About two days after emergence as adults most of the male and female bugs copulate for the first time. A copulatory attempt consisted in the male running to chase the female, and if the female does not reject the male, by running away from the male, the male jumps on the back of the female, and slipping to either the right or left side. Generally, the male mounts the female from the right side, but occasionally, it was observed that males would mount and cling from the left side. While both sexes are facing.in the same direction, the 8th abdominal segment and the pygophore of the male rotate slowly and phallus begins to erect in the act of engagement, the female lowers the valvulae, allowing the distending phallus to pass into the intervalvulae 11. space, which is inserted into the female almost up to the phallot\eca.
The male then swings around and the copulating pair assumes the final position of coitus, end to end, facing in opposite directions.
Close observation of the cotton stainers in the final position of copulation revealed that the 8th abdominal segment of the male had rotated 90° and the pygophore about 180°.
Sometimes the attempt to engage was unsuccessful, in which case the male normally resumed the central riding position and made further attempts, which may be successful or not.
It .is a remarkable fact that once copulation has started the pairs remain engaged for 4 to 6 days, moving, eating and drinking, and even excreting in this state. The females lay eggs shortly after disengagement eventually occurs. Copulation starts again after each batch of eggs have been laid. Therefore it can be said that young adult cotton stainer bugs are almost permanently engaged; though in the later part of their sexual life it may be observed male and female disengaged for longer periods of time. 29
With isolated pairs, it was noted that if the male died prematurely after starting to copulate the female might lay, generally, only one batch of fertile eggs; the following batch would be of sterile eggs, suggesting that sperm is being continuously transferred over long periods of normal copulation.
A microscopic examination of the terminalia of the engaging pairs either after boiling in KOH, or from .vivisected material, showed that the vesica is in a vertical position with the secondary gonopore applied to the opening of the female spermathecal duct. Therefore, the fluid which contains the sperm can be transferred directly to the spermathecal duct.
The vesica is maintained in that position by the pressure of the apex of the pygophore against the 9th gonocoxite of the female, on the back side, and laterally by the processus conjunctivae. The phallus is supported, ventrally, by the 11th abdominal segment (anal tube) that is fixed against the constriction between the phallotleca and the conjunctiva. ge•
opening, There is a membrane surrounding the secondary gonopore to facilitate adhesion between the vesica and the sclerite present around the opening of the spermathecal duct. 30
SECTION B. THE EFFECTS CF SOME POTENTIAL CHEMCSTERILANTS
INTRODUCTION
In the last decade the use of chemosterilants for insect control has become a possible alternative to radiation mass- reared programmes for the release of sterile males. They cost less and they provide the most practical tool for sterilization of insect pests in large number.
Chemosterilants may be used as a direct treatment and the effectiveness of direct spray application on cotton plants was shown by McGovern et al. (1969), demonstrating the potential of the method should safer chemicals be eventually discovered. They could also be used to treat selective resting places, or in conjunction with chemical attractants in baits, in order to minimize hazards, although even the advisability of this approach has been questioned by Ascher (1967), since the most effective chemosterilants are hazardous to man and other animals. They are mutagenic in acute doses.
The activities of the chemosterilants or insects have been reviewed by Smith et al. (1964) and Campion (1965, 1972).
Borkovec (1966) described the chemistry of chemosterilants and their application; LaBrecque and Smith (1963) reviewed the potential, chemistry and application of chemosterilants.
It must be emphasized from the beginning that the examples given in this thesis will be as far as possible restricted to bugs; and a comprehensive survey is considered to be beyond the scope of this study. Cheillosterilants can be categorized as follows: 31
Alkylating agents
Alkylating agents were the first group of chemicals found
to sterilize insects and they include aziridines, nitrogen mustards
and sulphonic acid esteres according to their functional groups
(Ross, 1962).
The sterilizing activity, in this group, against male
insects may result from the alkylation of the nucleic acids of
the mature sperm nucleus, whereas in the females the mode of
action of biological alkylating agents is uncertain (Borkovec, 1966).
Tepa (tris (1-aziridinyl) phosphine oxide) is included in
the aziridine class and is one of the chemosterilants considered
in this study; this class also includes apholate, metepa and
thiotepa. Tepa has been used on a variety of insects, in the
laboratory and also in a few field experiments, with varying
degrees of success in the induction of sterility (Fig. 3).
Complete sterility following treatment with tepa has been
reported, mainly in Lepidoptera (Young and Cox, 1965; Topozzada
et al., 1966; Hathaway et al., 1966 and 1968; Campion, 1967 and
1970; Cox et al., 1967; Harding, 1967; Kishaba et al., 1967;
Flint et al., 1968; Campion and Lewis, 1971); and in Coleoptera
(Hedin et al., 1964 and 1967; Ladd, 1968; Ladd et al., 1968
Malik, 1970; Ondracek and Matolin, 1971). In some insects such
as alfafa weevill, Hypera postica (Gyllenhal) recovery of fertility
occurred (McLaughlin and Simpson, 1968).
Carcavallo and Carabajal (1971) carried out sterilization
experiments with tepa against three species of Triatominae.
The chemosterilant was applied topically to the fifth nymphal
instar and alsr, to thee atiu,,lt stage. All species proved sensitive 32
0 112C
,,N- p -N
H2 C s I C H2 H /\1\T
2C CH 2
Fig. 3. Structural formula of tepa (tris(1-aziridinyl) phosphine oxide. 33
to the treatment, but the best results were obtained when nymphs were treated, which resulted in 78 - 80% sterility as compared with 56 - 70% sterility when adult males were treated.
Males of the large milkweed bug fricopeltsciaLus were completely sterilized by topical application and injection of tepa and metepa. Tepa at 50 nliter caused 100% mortality four days after treatment, but metepa was a relatively non toxic sterilant at 1000 pg/kg (Economopoulos and Gordon, 1969).
Gomez-Nunez (1971) described the results obtained when males of Rhodnius prolixus were treated topically with metepa. Complete sterility was approached with 0.10 mg of metepa per male and induced oviposition became significantly lower with larger doses.
Mustafa and Naidu (1964) reported that the males and females of the red cotton bug Dysdercus cingulat-,zs were sterilized when exposed to surfaces on which apholate was deposited and that the degree of sterility was a function of the concentration of the chemosterilant and time of exposure.
Antimetabolites
This group includes analogues of folic acid, glutamine, purine and pyrimidine. Their action is more pronounced against females than against males (Borkovec, 1966), and they act by interfering with the synthesis of nucleic acids.
Masner and Macha (1968) and Mocha (1969) reported that
6-azauridine, orally administered, affected ovarian growth and prevented the synthesis of juvenile hormoue from the corpus allatum of the lime bug arrhocoris apterus Masner and Landa
(1971) showed that when 6-azauridine was supplied to P. apterus females in 0.001 - 0.1% water solutions in cotton, complete 34
sterility was achieved at the lower concentrations, while at higher concentrations, oviposition was completely inhibited.
Phosphoramides and s-triazines
Hempa, tris (dimethylamino) phosphine oxide is a highly effective chemosterilant, but according to Borkovec (1966) is less species-specific than, many s-triazines. Its physiological and cytological properties resemble the effects of alkylating agents- (Morgan, 1965). However when hempawas applied topically to males of Oncopeltus fasciatus it was shown to be inactive, even at a dose of 1000 mg/kg (Economopoulos and Gordon, 1969).
Chemosterilants derived from s—triazines comprise aziridinyl-s-triazines (tretamine); melamines (hemel); and diamino-s-triazines (Borkovec et al., 1972) (Fig. 4).
Chemosterilants of the s-triazine group have received much• attention in recent years; results of experiments with active aziridinyl-s-triazines and melamines were published (Borkovec and Terry, 1965; Borkovec and DeMilo, 1967; Borkovec et al., 1968
LaBrecque et al., 1968), and effects of 2, 4-diamino-s-triazine in the house flies are reported by Borkovec et al., 1972.
Economopoulos and Gordon (1969) reported that tretamine, applied topically, completely sterilized males of the large milkweed bug Oncopeltus fasciatus, with little adverse effects on longevity, spermatogenesis or mating, but the sterility persisted only for the first two .weeks after application.
After this period some males showed a partial recovery of fertility. Adult females of the same species wero also sterilized when treated topically with 1 jig of tretamine (Economopoulos, 1971). 35 Fig. 4. Structural formula of s-triazinesused in the experiments. (a)- N, N', N", trimethylmelomine;
(b) Pontamethylmelomine; (c)- Diamino (2-fury1)-s-triazine. 36
/C H 3
(a) /\ N N
N H3 C/ \ CH3
.C1-13 N/ (b) /C\ N
H3C il
\Nit H3C \CT-I 3
CH
CH
(z) N Ri
•RI 37
Economopoulos (1971) studied the effect of tretamine on 4th and early 5th instar nymphs of the large milkweed bugs and observed that this chemosterilant almost completely prevented the next moult while a small percentage developed into abnormal adults. When treatment was administered on mid or late 5th instar nymphs however there was no apparent effect on moulting to adults, suggesting that treatment at the late stages was not critical for this moult, since the epidermal cells had already differentiated.
Miscellaneous compounds
This group includes chemicals apparently unrelated, but which have shown sterilizing activity when applied to insects
(Borkovec, 1966; LaBrecque, 1968; Campion, 1972). The adult milkweed bug Oncopeltus fasciatus is highly sensitive to the effects of 2-imidazolidinone, an urea derivative. Dosages as low as 0.005 % given in water to nymphs inhibit development, and adults fed on the same concentration laid eggs, which did not hatch. Bugs injected as either 5th instar nymphs or newly emerged adults or when provided with water containing 0.5%
4-imidazolin-2-one for up to five days, resulted in the inhibition
of development and of the reproductive organs (Simkover, 1964;
Schaefer and Tiernan, 1967).
Insect hormones and analogues,
Hormones are internal secretions that regulate a wide
variety of physiological processes and especially those that
result in growth, development and maturation of insects. Two
of these are juvenile hormone and moulting hormone. TABLE 2. A survey of the chemosterilant activity of alkylating agents, aziridine derivatives, phosphoramides and s•-triazines on nymphs and adults of Hemiptera (Heteroptera).
Family Species name Common name Chemosterilant Stadium and Method of Activity Reference sex treated application
Pyrrhocoridae Cncopeltus Large milk- Tepa Males topical & + + Economopoulos & fasciatus weed bug injection Gordon (1969) Triatoma Tepa Nymphs topical + + Carcavallo & guasayana Carabajal(1971) Males & topical females Triatoma Tepa Nymphs topical + + Carcavallo & infestans Caraba jal(197:L) Males & topical females \.,74 Triatoma Tepa Nymphs topical + + Carcavallo & oo patagonica Carabajal(1971) Males & topical females
Pyrrhocoridae Cncopeltus Large milk- Metepa Males topical & Economopoulos & fasciatus week bug injection Gordon (1969) Reduviidae Rhodnius Reduviid bug Metepa Males topical + + Gomez-Nunez(1971) prolixus Pyrrhocoridae Dzsdercus Red cotton Apholate Males & contact + + Mustafa & Naidu cinf;ulatus bug females (1964) Cncopeltus Large milk- Mono-& bis- Males topical & fasciatus week bug aziridinyl injection Tretamine Nymphs topical + + Economopoulos(1971) Tretamine Males topical & + + Economopoulos(1971) injection Hempa Males topical & Economopoulos & injection Gordon (1969) * ft + + = active; + = slightly active.
Mi Vvo TABLE 3. A survey of the chemosterilant activity of antimetabolites, juvenile hormones and other products on nymphs and adults of Hemiptera (Heteroptera).
* Family Species name Common name Chemosterilant Stadium and Method of Activity Reference sex treated application
Pyrrhocoridee arrhocoris Lime bug 6-azauridine Females Oral + Masner & Macha apterus (1968) 6-azauridine Females Oral + + Macha (1969)
6-azauridine Females Oral + + Masner & Landa (1971) "Paper factor" Nymphs Contact + + Slama & Williams (1966) DDIF (1) Females Topical + + Masner et al. (1968) DMF (1) Eggs & females Topical + + Matolin (1970)
Dysdercus Cotton DEF (2) Adults + + Topical Bransby & cerdinalis stainer bug Williams(1971) Dysdercus Cotton DMF (1) Nymphs & Topical + + Critchley & fasciatus stainer bug both sexes Campion (1971) DMF (1) Nymphs & Topical + + Campion & females Critchley(1972) Oncopeltus Large milk- 2-imidazolidi- Nymphs & Oral & + + Simkover(1964) fasciatus weed bug none males injection 4-imidazolin- Nymphs & Oral & + + Schaeffer & 2-one males injection Tiernan (1967) Juvenile (3) Nymphs Topical + + Bowers (1969) hormone (VII) "Paper factor" Nymphs Contact + + Carlisle & Ellis (1967) (1)Methyl farnesoate dihydrochloride. (2)Diethyl farnesoate dihydrochloride. * - + + active« (3)(6,7- epoxy -3,7- dimethyl-oct-2-enyl ether of p-carbomethoxyphenol). 140
The juvenile hormone, in addition to controlling the expression of larval characteristics and inhibiting metamorphosis, is concerned in the reproductive activity of both sexes; it is related to simple plantterpenes.
Moulting hormone constituents are steroids, the chemical structure of which are now known, and ecdysones have been isolated and crystallized in pure form from larvae of Calliphora and from pupae of the silkworm. The hormone exerts effect directly upon these cells concerned with growth and moulting..
The results of many different experiments suggest that juvenile liormone is produced by the corpus allatum and eodysone
(moulting hormone) by the prothoracic gland. These two hormones are largely responsible for the development of the insect from the egg, through hatching, growth and several moults to the adult (e.g. review by Novak, 1966).
It was in 1956 that Williams first suggested the use of insect hormones to provide the basis for a different method of insect control. The basic idea behind the use of hormones for insect control is to exploit these processes so that death or sterility occurs, by applying the hormone at a time when it would normally be absent or would be present in only very small amounts (Ellis, 1968). These substances are almost certainly not hazardous to mammals.
The possible use of these two hormones and their mimics as pesticides for insect control has been reviewed, amongst others, by Williams, 1967; Mordue, 1969; Ellis et 31., 1970.
Hormones control certain stages of spermatogenesis and oogenesis (Clark and Langley, 1963). For example it has been found that some juvenile hormone analogues, which are related 41 chemically to farnesol have sterilizing effects on the female
Pyrrhocoris apterus, after topical application, resulting from inhibition of embryonic development (Slama and Williams, 1966;
Matolin, 1970).
Topical application of methyl farnesoate dihydrochioride
(DMF) to females of the lime bug P. apterus, any time during the reproduction cycle, caused permanent sterilization (I4asner et al.,
1968). It was also noted that the insect tolerated doses of up to 10,000 times higher than the sterilizing dose, without any adverse effects.
Bowers (1969) reported a very high activity of juvenile hormone analogue VII (6, 7-epoxy-3, 7 dimethyl-oct-2-enyl ether of -p-carbomethoxyphenol). which inhibited metamorphosis in
Oncopeltus fasciatus at doses below 10 vg.
Matolin (1970) confirmed that DMF was a potent juvenile hormone analogue, when applied topically to females of P. apterus, and showed that it induced abnormalities in embryonic development which resulted in the inhibition of egg hatch.
Critchley and Campion (1971) treated topically 3rd, 4th and
5th instar nymphs of the cotton stainer D. fasciatus with DMF.
Some nymphal treatments resulted in adults incapaole of reproduction and others produced abnormal forms, such as sixth- instar nymphs, juvenile adults and super-adults. Similar effects to those noted for P. apterus, were obtained by Critchley and
Campion (1971) when adults of ysdercus fasciatus were treated topically with DNF; complete sterility wc.?y achieved at a dose of 5 Kg for females and 50 pg for males.
The insect moulting` hormones and analogues have been found to inhibit ovarian maturation and egg production when fed to
e Adult females of 42
Anthonomus grandis when fed ecdysone analogue were permanently
sterilized while the males were not affected (Earle et al., 1970).
It is concluded from this survey of the chemosterilization
of Hemiptera (Heteroptera), which is also summarized in Tables 2
and 3, that there is very little information concerning the
effect of chemosterilants on nymphs or adults of Dysdercus
fasciatus Signoret, although information concerning some other
species of Heteroptera is available.
For this reason, an aim of the present work was to provide
an evaluation of the actions of selected potential sterilants upon
D. fasciatus, in order to consider the possibility of using a
suitable chemosterilant in the field. Since from a theoretical
point of view it is generally assumed that the male insect is the
more appropriate target in chemosterilization, most emphasis in
this thesis is given to the effect of chemosterilants on the male cotton bugs; however it is necessary to point out that release of
both sexes of sterile insects was used for the successful
eradication of the screw-worm from the SoUth-eastern United
States (Knipling, 1958).
It has been found that moulting in Oncopeltus fasciatus was
prevented almost completely when chemosterilants were applied at
the beginning of the 4th- and 5th- nymphal instars (Economopoulos,
1971). For this reason the effects of chemosterilants on the
nymphal stages of D. fasciatus were also investigated. 43
MATERIAL AND EEIHODS
1. Experimental methods.
1.1. Method of immobilization. Just before treatment, the insects were transferred to a deep freeze at - 15°C for 3.5 - 4.0
minutes. This procedure immobilized the insects for up to 5
minutes after they were returned to room temperature. This was
a sufficient time to treat a batch consisting of not more than
15 insects. No deaths by freezing occurred during all the
experiments and the insects soon recovered without any apparent
deleterious side effects. Controls were also immobilized in
this way before being treated with 0.6 p1 cf acetone.
1.2. Method of topical ap lication. Microdrop quantities
of the various chemosterilants to be tested were applied topically
to the nymphs and male adults on the ventral surface of the
abdomen and towards the apex with a Burkhard micro-drop applicator
fitted with a one ml Agla-all-glass syringe and a 20 G needle.
Nymphal stages were treated topically with doses of tepa
ranging between 0.1 and 15 pg on 3rd instar and 5 and 50 pg per
nymph on 5th instar.
For the experiments on sterility doses between 0.5 and 5.0 pg
of tepa and between 3 and 24 pg of s-triazines were applied to
males in 0.6 pl of acetone. For toxicity tests doses between
103 and 206 jig, in 2 x 1 p1 of acetone, were applied per male.
1.3. Residual film technique. For residual treatments,
quantities of tepc were dicsolved in acetone, ond 4 ml rill aunts
of solution were poured into the bottom of Monax petri dishes 2 with an area of 78.5 cm . After solvent evaporation an even 44 film of the chemosterilant was deposited in concentrations ranging from 0.5 to 15 pg/cm2.
To allow for solvent evaporation the dishes were stored overnight in a fume cupboard. The following morning young males, up to 48 hrs old, were selected and divided into a number of batches, each of five insects. Each batch of insects was exposed to the residual film for two hours.
Control dishes were treated with 4 ml of acetone and cotton stainer bugs confined in a similar manner.
The sides of the petri dishes were painted with a fine film of silicone fluid (F111/20, ICI) to ensure that the cotton stainers remained on the treated surfaces.
1.4. Post-treatment procedure. After treatment, cotton bug pairs, consisting of one treated male and one untreated virgin female of the same age, were placed in plastic sandwich boxes,
19 x 11 x 8 cm with a substracte of damp peat. These boxes were held in a constant temperature room at 27+0.5°C and 70 4, 5% relative humidity, with a 12 hours photoperiod under artificial light. All the plastic boxes were painted around the rim with a fine film of silicone fluid, to prevent the insects escaping.
Every second day all the eggs were collected from each box and each batch was incubated separately, until they hatched, in small petri dishes containing soaked and squeezed peat. Records were also kept of the number of eggs laid per batch and from each female, and also the number of 1st instar nymphs that were obtained after seven days of incubation. From these figures the effect of treatment on fertility was calculated. 45
At the time when the soaked cottonseeds and soaked cotton wool, which constituted the insects diet and the available drinking water, were changed, an assessment of mating ability was made by observation of the undisturbed paired bugs and a record was kept on whether or not copulation was in progress.
At the end of each experiment the total number of "mated" days for treatments and controls was then compared.
In the experiments with nymphs daily examinations were carried out for mortality or for any abnormality that might have occurred during development. Adult males and females, surviving from the nymphal treatments, were caged with normal insects of the opposite sex of similar age, receiving identical treatment as previously described for treated males. A period of four weeks after pairing was judged sufficient to see whether an adult, resulting from a treated nymph, was able or not to reproduce. After this period adults were dissected to determine the presence or absence of sperm and its motility in the testes and in the spermatheca, and also to detect other possible abnormalities.
2. Estimation of the median sterilizing and lethal doses.
2.1. Sterilizing doses. In order to determine a dose- response relationship for the sterilizing effect of tepa on adult males of D. fasciatus, graded doses and concentrations of the chemosterilants were used in the manner previously described.
All the chemosterilants solutions used were made from the stock solution in acetone, stored in the deep freeze. 46
2.2. Lethal doses. Male adults of D. fasciatus, up to
48 hrs old, were treated with graded doses of tepa, applied topically, to determine the mortality effect of this chemosterilant.
The effects on mortality were recorded for up to three weeks after treatment.
3. Statistical methods.
The percentages of egg hatching, in the control and in the different treatments were compared by Chi-square tests, these were used when it was necessary to compare results with only a single degree of freedom (2 x 2 contingency Gables).
The data from each biossay were analysed by the London
University CDC 6600 Computer, using a programme for prObit analysis developed by the Canadian Department of Agriculture.
For each set of results slopes, heterogeneity, ED50 and fiducial limits were obtained.
4. Chemosterilants and solvents.
The solvents used were acetone (Analar) for tepa and a mixture of acetone (Analar) and dimethyl sulfoxide (DMS0) (7:3) for the s-triazines.
The following chemosterilants, provided by Dr. A. B. Borkovec and obtained from Dr. D. G. Campion, were tested for sterilizing effects on Dysdercus fasciatus :
a) Tepa (tris (1-aziridinyl) phosphine oxide). Stock solutions at a concentration of 103 mg/ml were prepared.
b) Diamino (2- fury1)-s-triazine .
c ) N", trimethyl m els mine. d ) Pentamethylmelamine.
Stock solutionscontaining 40 mg/m1 were made up of the
three r3-triazinAs, in acetone: DMSO (7:3). 1+7
Tepa was one of the chemosterilants chosen because its wide spectrum and efficiency against many insects; and the s-triazines tested were used to compare their effectiveness with tepa, since there was little information of them as insect sterilizing agents and would be interesting to know the results that could be obtained when D. fasciatus would be treated with these s-triazines (A. B. Borkovec, private communication). 48
1-117.C.TTTMO 11.1:JOUL10.
1. Teatreatmentson3rclinsta/ihsbtoicala15p •n ppk_cation.
The results obtained in this experiment showed that when young
3rd instar nymphs were treated with progressively increased doses
of tepa in acetone, the number of nymphs that moulted into 4th
and 5th instars progressively decreased; as did the number of
surviving adults (Table 4 and Fig. 5). In these effects the test
insects differed significantly from the controls at doses of 10 Fs
and above per nymph (P < 0.001) • For example only 8.8% of the
nymphs treated with the higher dose of 15 pg/nymph, turned into
adults, compared with 67.9% in the control. Fig. 5 shows that all the nymphs treated at the 3rd instar stage were affected by
tepa in their subsequent development.
Tepa treatment also affected the proportion of mated pairs
able to reproduce. Significant differences when compared with
the controls were noted when doses of 10 pg per nymph was applied
(P=0.025), while at a dose of 15 .Fg per nymph oviposition was inhibited, although at that dose in fact only 5 adults were obtained from 57 treated nymphs (Table 4).
batches of oggs in relation to treatment Guggor,ted that 14ith
1. 1
of the fomni^!:.
TrChTn 5 f71ow7, the rnr4111t of thn (ii rAr.leeti_onrt mndr, on ster5le
adults which were treated as 3rd instar nymphs. It can be seen
that only 4 of the 42 females had motile sperm in the spermatheca,
but 38 of the 42 males had motile sperm in the testes and the vesicula seminal? s, 49
TABLE 4. The effect of tepa on nymphal development and on reproductive potential, when applied topically to early 3rd instar nymphs. Surviving adults were paired with normal virgin adults of the opposite sex.
Dose No. laryiziELL1LILLAdults able to reproduce Mating Batches pg/ nymphs Corr- Pairs days* per pair* nymph treated No. % ected mated No. /0 % (1) (2) (2:1)
15.0 57 5 8.8 13.0 5 0 0.0
10.0 56 19 33.9 49.9 17 2 11.8 3.8 2.0 1.0 55 33 60.0 88.4 33 13 39.4 3.6 1.9 0.1 57 37 64.9 95.6 36 16 44.4 3.0 1.5
TOTAL 225 94 41.8 91 31 34.1
CONTROL 56 38 67.9 100.0 34 15 44.1 5.6 4.6
* - Average for fertile pairs. 14148.1-*-4 4 .1.9es WAAL W) C9"411 •
TABLE 5. Occurrence and condition of sperm in the spermatheca and testes of mated but sterile pairs, when 3rd instar nymphs were treated with tepa.
Sterile females Males Dose Number Pg/ No Non- Motile No Non- Motile of nymph sperm motile sperm sperm motile sperm pairs sperm sperm
15.0 0 1 0 0 0 1 1
10.0 2 1 1 0 0 4 4
1.0 8 3 2 0 0 13 13 0.1 9 4 0 0 1 12 13
TOTAL 19 9 3 0 3 30 11
CONTROL 7 3 1 0 4 8 42 50
DOSE pginymph
e____ Control
A------0.1
1.0
10.0
. 15.0
3rd 4th 5th Adults
IN STA II S
Fig. 5. Effect of tepa on nymphal develop- ment when applied tcpically as early 3rd instar nymphs. 51
2. Tepa treatmentssajth instar nymphs,by topical application.
The results showed in Table 6 suggested that there was a direct relationship between doses applied and the number of nymphs that died before moulting, when these nymphs were treated topically with tepa at the early 5th instar. This relationship is expressed by the following regression equation (Fig. 6 and Table 12):
y = 3.65 0.78x
The percentage of treated nymphs that became adults was shown to be significantly different, by Chi-square test, from the corresponding percentage of nymphs treated as controls with acetone only (P<0.001)• This result showed that tepa treatments disturbed the normal development of the early 5th instar nymphs of D. fasciatus. Although surviving adults resulting from treated and control nymphs were always able to reproduce to some extent, it is necessary to emphasize that the number of batches of eggs
per female was reduced in females resulting from treated nymphs when compared with controls (e.g. females from nymphs treated with
50 jig of tepa produced 1.1 batch and controls produced 2.1 batches).
It may be worth noting that the number of batches of eggs per female in the control was itself low compared with other strains used in later experiments; i.e., the strain of insects that was used in the
chemosterilant experiments was later revealed to be rather poor.
When nymphs were treated with different doses of tepa, late in the 5th instar, the number of nymphs that moulted to the adult
stage was not significantly different from the controls (P=0.3).
It can therefore be concluded, for the purposes of this experiment,
that 2 - 4 days after moulting to fifths the nymphb were not
affected by tepa treatment over the range of doses tested(Table 7). 52
TABLE 6. Effect of tepa on nymphal development and on reproductive potential, when applied topically to early 5th instar nymphs (0 - 48 hrs. after moulting). Surviving adults were paired with normal virgin adults of the opposite sex.
Dose No. survim_Tizadul ta 2121- 2 I2...E221a:da22 Mating Batches nymphs Corr- Pairs days* per nymph treated No. % ected mated No. pair* (1) (2) (2:1)
5o 52 22 42.3 47.0 23 8 34.7 2.9 1.1
25 54 32 59.3 65.8 32 12 37.5 3.5 1.5 lo 55 35 63.7 70.7 33 12 36.3 3.3 1.4
5 53 37 69.8 78.9 32 15 47.2 3.0 1.8
TOTAL 214 126 58.8 65.6 120 47 39.1 3.2 1.4
CONTROL 50 45 90.0 100.0 34 16 47.1 3.4 2.1
TABLE 7. Effect of tepa on nymphal development and on reproduOtive potential, when applied topically to late 5th instar nymphs (2 - 4 days after moulting). Surviving adults were paired with normal virgin adults of the opposite sex.
Doge No. Survivin adults Adults able to reproduce Mating Batches lig/ nymphs Corr- Pairs days * per nymph treated No. % ected mated No. pair* (1) (2) (2:1)
50 45 38 84.4 92.0 38 22 57.9 3.6 1.7 25 45 38 84.4 92.0 34 21 61.8 3.8 1.h 10 47 39 83.o 90.5 29 16 55.1 3.6 2.0 5 45 36 80.o 87.2 29 20 68.9 3.2 1.6
TOTAL 182 151 82.9 90.4 130 79 60.7 3.5 1.7 CONTROL 36 33 84.4 loo.o 3o 20 66.6 3.6 1.8
. * Average for fertile pairs. Nv•v".(m 4 LT? vut 'En 100 O. 50 Fig. 6. I- 0 .■ = 0 0 C m. 0 c); Fig. 7. mort a lity 4- '
Probit regressionlineformortality treated topicallywithtepa. of 5thinstarnymphsD.fasciatus 0.5 surviving adults whennymphs were Influence oftopeonthenumber of treated asearly andlate 5th instar. Early inster
LOG DOSE(pg/nymph) O o 0 1.0 o O
57
1.5 Late instar
0 0 O 0 0 0 0 2.0 0 0 0 0 0 0 0 a U a a ❑ 0 ug/ nymph Control DOSE 50 25 10 5 54
This difference with age was confirmed by comparing the total numbers of tepa treated nymphs that eventually became adults, when they were treated as early and late 5th instar (Tables 6 and 7).
These numbers were significantly different by Chi-square test (P<0.001).
TABLE 8. Occurrence and condition of sperm in the spermatheca and testes of sterile pairs, when 5th instar (0 - 2 days old) nymphs were treated.
Dose Sterile females Males Number of Pg No Non- Motile No Non Motile pairs nymph sperm motile sperm sperm motile sperm sperm sperm
5o 6 2 0 0 2 6 8
25' 4 1 1 1 0 5 6
10 11 1 0 3 1 .8 12
5 11 0 0 3 2 6 11
CONTROL 10 3 0 1 2 10 13
TOTAL 42 7 1 8 7 35 50
TABLE 9. Occurrence and condition of sperm in the spermatheca and testes of sterile pairs, when 5th instar (02- Vidays)
nymphs were treated.
Dose Sterile females Males Number of
Ile No Non- Motile No Non,- Motile pairs nymph sperm motile sperm sperm motile sperm sperm sperm
50 3 2 1 4 5
Pc 4 2 1 1 6 7
10 8 1 0 1 5 3 9
m 9 3 3 1, G 12
CONTROL 9 1 0 5 10
Mt,MAT If 12 .71-1 46 JJ 7 4 55
Thus tepa was shown to have affected the development and the reproductive potential of nymphs aged up to two days after moulting, at the time of treatment, but had no observable effects when applied to nymphs at an age of 2 - 4 days after moulting (Tables 6, 7 and
Fig. 7).
Considering the results of the dissections on the females and males of sterile pairs, presented on Tables 8 and 9, it was evident that in the majority of these females no sperm was present in the spermatheca; a few possessed non-motile sperm, while a very few sterile females had motile sperm in the spermatheca. On the other hand a high percentage of males contained motile sperm in the testes and vesicula seminalis, although this sperm was evidently not transferred to the spermatheca of the female insects. It would be concluded that sterility of nymphal treated insects was largely correlated with failure to transfer sperm even though pairing occurred normally.
3. The effect of tepa on adult males, applied topically. The results of the dose-response data observed (Tables 10,11 and
Fig. 8) showed that there was a linear relationship between the log doses and the percentages of induced sterility and mortality
The SD and LD values of the topical expressed as probits. 50 50 applications of tepa are given in Table 12.
A test of parallelism was carried out for both regression lines, sterilizing and mortality effects, and it was found that the data did not contradict the hypothesis of parallelism. It was therefore possible to calculate the sterility index, that is the ratio between lethal and sterilizing doses of tepa to Dysdercus adult males. As the sterilizing dose (SD50) was 0.8792 pg and the lethal dose (LD50) 56
221.5 pg per male, the sterility index was very high (251.9).
This indicated that a wide safety margin existed between these two doses. It would be expected that this lack of toxicity would allow treatment of males with permanent sterilizing doses without reduction of mating vigour. On the other hand, the degree of selectivity of chemosterilants for gonadal tissue, that was calculated by the ratio LD50/SD90 is equal to 56. This very high value indicated a high degree of selectivity of tepa for gonadal tissue of D. fasciatus (Chang and Borkovec, 1966;
Turner, 1968).
Over the range used in sterility tests different doses of tepa did not have any effect on the number of mating-days, the number of egg batches or on the rate of oviposition per female, with the exception of the highest dose per male, which was 5 pg.
This dose level reduced the number of egg batches and the fecundity per female by approximately 40 and 60% respectively, when compared with the controls, probably at this dose some tepa might be transferred to the females by contact with males during copulation since mating takes place just after the males being treated with males.
Counts were made of both the number of eggs and the number of
1st instar nymphs that subsequently emerged from each clutch of eggs. This made it possible to assess whether there was an alteration in the pattern of fertility of all batches, as a result of the different doses of tepa applied. This effect is shown in Fig. 9.
All the doses caused an immediate reduction in hatch, which was proportional to the doses applied. At a dose of 2.5 pg/male, for example, there was almost complete sterility in the first three batches of ecgs, but subsequently a progressive recovery in fertility occurred. Complete sterility of all clutches of eggs was observed at 3 minimum 5,n pg/male. 57
TABLE 10. Sterilizing effect of tepa when applied topically, to male ysdercus mated with untreated females.(I)
Dose Mating Batches Fecundity Eggs Fertility Sterility Corrected pg/ days per per hatched 0//0 0/p sterility male female female per 0//0 female
0.5 11.0 6.5 405 173 42.7 57.3 39.2 1.0 9.o 6.o 364 132 36.3 63.7 48.4 1.6 8.3 7.0 319 77 24.1 75.9 65.6 2.5 13.5 8.o 46o 64 14.9 85.1 80.2 5.o 6.5 4.o 164 0 0.0 100.0 100.0
=ROL 8.2 7.0 393 276 70.2 29.8 0.0
(1) Results from a minimum of five pairs, for each dose, but excluding incompatible pairs.
TABLE 11. Toxicity of tepa to adult males of D. fasciatus, tt„ mmaks „ILA buari0AA,,r.
Dose No. males No. males Mortality Corrected 0/ pg/male treated dead /0 mortality
103 32 5 15.6 15.6 137 33 9 27.3 27.3 165 34 13 38.2 38.2 206 36 16 44.4 44.4
CONTROL 32 0 0.0 0.0 58
Sterility a y=3.15i•1.96x
Mortality y=-1.854-2.92x
1
1.0 1.5 2.0 1.5 2.0 2.5
LOG DOSE (pg/male x10 ) LOG DOSE (pg/male)
Fig. 8. Probit regression lines for sterility and mortality induced by tepa against male D. fasciatus, when treated topically. 80
pgirriale
Control
0.5
40 1.0
1.6
2.5
5.0
0 D 0
1 2 3 4 5 6 7
Batches of eggs
Fig. 9. Effect of tepa on the pattern of fertility of D. fasciatus, during the oviposition period, when males were treated by topical application, compared with controls. bo
4. The effect of tepa on the nymphal stage in the F generation. 1 To discover whether tepa treatments of adults had any effect on the nymphal stage of the progeny from P1 male, batches of eggs were collected from treated pairs (treated male x normal female), and their development was followed until they reached the adult stage (Table 13).
It can be seen that the number of surviving adults from treatments was significantly lower than in controls (P <0.001), as the result of a high mortality of nymphs that occurred mainly when moulting from 2nd to 3rd instar and from 5th instar to adults, but considerably higher in the former case.
Therefore, the treatment of male parents affected the development of the subsequent progeny, when these males were paired with normal females. A small percentage of abnormal adults, with distorted wings was also observed, but only when they resulted from nymphs from male parents which had been treated with tepa.
5. Residual action of tepa on male of Dysdercus.
Batches of ysdercus males were exposed for two hours at 2 concentrations of tepa, varying between 0.5 and 15.0 pg/cm , deposited as a residual film. No mortality was observed in any of these concentrations, after treatment.
The results, which are summarized in Table 11F, showed that sterility was induced in males of D. fasciatus as the result of treatment and, also, that the degree of sterility, as in the case of topical applications, varied with the concentration of tepa. 2 Thus, a concentration of 0.5 pg/cm induced only 10.2% of sterility; 2 at a concentration of 10.0 pg/cm 95.4% of sterility occurred, while 15 pg/cm4 induced complete sterility. 61
TABLE 12. Probit analysis data for sterilizing and mortality effects of tepa against 5th instar nymphs and male adults of D. fasciatus.
Method of Parameters Heterogeneity 95% fiducial application of probit _ _ limits lines x y ED .,2 50 a b+s.e. X (d.f.) P jig/ Lower Upper male Topical application Sterility regression for male adults 5.11 1.96+0.37 .1382 5.3797 17.63(3) <0.001 0.8792 0.35 1.3 Toxicity regression for 5th instar nymphs 3.65 0.78+0.28 1.2588 4.6349 0.88(2) 0.60 53.07* 27.34 831.4r Toxicity regression for male adults 4.85 2.92+1.07 2.1884 4.5415 0.19(2) 0.90 221.50 179.80 595.00 Residual film Sterility regression for male adults 4.32 1.99+0.42 .5028 5.3199 56.28(4) <0.001 2.20** 0.99 3.36
* - iig/nymph ** pg/cm2
TABLE 13. Effect of tepa on the progeny of male Dysdercus treated topically and crossed with normal virgin females.
Dose Number of nymphs on different instars Nymphal period Pg/ Adults surviving male 1st 2nd 3rd 4th 5th adults (days)
1.6 59 58 34 33 33 32 54.4 26 - 28 o.8 149 148 89 87 85 74 49.7 25 - 27 0.4 125 119 78 72 69 58 46.4 24 - 27
CONTROL 98 95 77 77 71 72.4 24 - 27 62
The SD value for the residual film method is given in 50 Table 12 and the probit line is illustrated in the Fig. 10.
Data from Table 14 showed that tepa treatment, by residual
contact, to male Dysdercus, had no apparent effect on mating and
the number of batches of eggs at all concentrations of tepa used, 2 but fecundity started to decrease at a level of 10 pg/cm .
In a manner similar to that following topical application,
tepa caused a reduction in egg hatch of each clutch of eggs (Fig.11).
At 0.5, 1.0 and 2.4 pg/cm2, the percentage hatch decreased after
a peak at the 3rd, 4th and 2nd batch, respectively. At 4.0 and 2 5.0 pg/cm the hatching percentage was very small, for the first
three batches, but subsequently some recovery of fertility occurred. 2 At the concentrations of 10.0 pg/cm egg hatching began only in
the 6th batch, thus the effect of this concentration lasted for
a period of more than three weeks, after treatment. Complete 2 sterility occurred at 15.0 pg/cm .
TABLE 14. Sterilizing effect of tepa on male Dysdercus, when applied by tarsal contact with a residual film(1)
Concn. Hating Batches Fecundity Eggs Fertility Sterility Corrected ci pg/cm2 days per per hatched % /0 sterility female female per /o female 0.5 8.5 0u 475 305 64.2 35.8 10.2 1.0 9.5 9 490 223 50.7 49.3 36.3 2.4 6.o 7 54o 210 38.8 61.2 45.6 4.0 12.0 9 625 180 28.8 71.2 59.7
5.0 9,0 8 472 56 11.9 88.1 83.4 n7 10.0 lu..t) 9 400 .,_, 3.5 or,-., r; 95.4 15.0 7.0 7 397 0 0.0 100.0 100.0 CONTROL 9.0 7 484 346 71.5 28.5 0.0 (1) Results from a minimum of five pairs, for each concentration, but excluding incompatible paira.
63
7
44 •ME
• IS 0
• NM 5 O O.
al=
3 —
0.5 1.0 1.5 2.0 2.5
L og concen, cm2 x 10 )
Fig. 10. Concentration/response regression for D.fasciatus males to the contact action of tepa. Concentrat i on 2 90 ig/c m Control
C.) a___ 0.5 0. • • 1.0 60 •
e • • • 2.4
tag • '0
n • • 0-- 4.0
rce • • e p • 5.0 • • . • ...... , Ca A- - -• - - - - -de a) ..- ... • 10.0 `
• • a-- 15.0
A 0 a t ----
1 2 3 4 5 6 7 8
Batches of eggs
Fig. 11. Effect of tepa on the pattern of fertility of D. fasciatus,from successive batches, when males were treated by tarsal contact, compared with controls. 65
6. Effect of s-triazines, when applied topically, to males of D. fasciatus.
The effects of treating male Dysdercus cotton bugs with graduated doses of trimethylmelamine and pentamethylmelamine on the fertility of untreated females to which they had been mated
and are presented in Tables 15 and 16. The calculated SD50 other probit data, for both melamines are shown in Table 17.
Trimethylmelamine and pentamethylmelamine were effective as chemosterilants against D. fasciatus, and 93.2% and 91.2% of sterility was obtained, respectively, at the highest dose used in the experiment, 24 jig/male.
From the data linear relationships were established between doses and sterility induced (Tables 15, 16 and Fig. 12).
The number of eggs from the treated pairs ranged between
146 and 231, for trimethylmelamine, and between 227 and 361, for pentamethylmelamine, while the mean in the controls was 411 eggs per female. The figures presented, suggested that s-triazines had a significant effect on the number of batches of eggs and on fecundity, and that this effect was most marked for trimethylmelamine.
From these data and from the two regression lines it was deduced that trimethylmelamine was the most effective of the s-triazines tested for the cotton stainer bugs, in spite of its higher SD value, 10.76 against 5.54 jig/male for pentamethylmelamine. 50 Trimethylmelamine also affected the number of mating-days, in comparison with controls. Competitiveness was therefore deduced to have been reduced; not a very good characteristic for a chemosterilant. 66 TABLE 15. Effect of trimethylmelamine on male Dysdercus, when applied topically.(1)
Dose Mating Batches Fecundity Eggs Fertility Sterility Corrected jig/ days per per hatched % 0//0 sterility male female female per % female
6 9.8 4.2 231 110 47.6 52.4 20.8
9 11.0 2.5 146 59 40.4 59.6 32.8 12 9.1 3.3 187 63 33.7 66.3 43.9 15 10.3 4.o 201 22 11.0 89.0 81.8 18 8.5 4.o 190 lo 5.3 94.7 91.2 24 8.8 3.6 197 8 4.1 95.9 93.2 co:ITROL 15.4 6.0 411 247 60.0 40.0 0.0
TABLE 16. Effect of pentametqi_melamine on male Dysdercus, when applied topically."-
Dose Mating Batches Fecundity Eggs Fertility Sterility Corrected 0/ Pe days per per hatched /L, % sterility male female female per % female
3 15.5 4.o 297 117 39.4 60.6 34.4 6 14.3 5.6 319 86 27.o 73.o 55.1 9 11.8 5.5 347 77 22.0 78.0 63.1 12 10.8 4.8 289 59 20,4 79.6 66.o 18 13.8 5.6 361 63 17.5 82.5 71.0 24 14.o 4.7 227 12 5.3 94.7 91.2
CONTPOL 15.4 6.o 411 247 6o.o 4o.o 0.0
Ali Roo -alto from o minim= of six pc:irs, =Dch dr)r4a, b't excluding incompatible pairs. 67
7 Trirnethylmelamine (0)
y=0.07 +4.78 x
5
Penta methyl melami ne ( co )
y=3.91+1.47x
3 1 1
0.5 1.0 1.5
Log dose (lig/male )
Fig. 12. Dose/response regression for D. fasciatus to s-triazines, when applied topically. 68
TABLE 17. Probit analysis data for the topically applied s-triazines.
Chemo- Parameters Heterogeneity 95% fiducial sterilant of probit — — limits lines x y SD 2 50 a b+s.e. X (d.f.) P fg/ Lower Upper male
Trimethyl- melamine 0.07 4.78+0.93 1.1389 5.5118 17.33(4) 0.002 10.76 7.27 12.99
Penta- methyl- melamine 3.91 1.47+0.29 .9956 5.3702 12.54(4) 0.016 5.54 2.35 7.96 69
Observing the patterns of egg hatch of the successive batches, for the two s-triazines, it was noted that for trimethylmelamine, the doses could be arranged in two distinct groups: up to 12 pg/male, with a maximum of 43.9% of sterility, and greater than 12 pg/male, with a minimum of 81.8% of sterility (Fig.13). For pentamethyl- melamine, there was no such distinction, with the hatching lines for different doses declining progressively, without a big transition (Fig. 14).
From all the results obtained, it was concluded that the melamines, used in these experiments, were quite good chemosterilants for males of D. fasciatus.
The effects of the potential chemosterilants against males of the cotton stainer bug afclercusfasciatus are summarized in
Table 18. It is clear that only tepa, when applied topically or by residual film, induced complete sterility at a dose of
5 pg/male and at a concentration of 15 pg/cm2. Trimethylmelamine was the more effective of the three s-triazines tested inducing
93% sterility, at a dose of 24 pg/male, but the number of mating days was reduced, therefore competitiveness would be also reduced
(Fig.12). Pentamethylmelamine was so effective at the same dose, but at higher doses the sterilizing effects were not so good
(Fig. 12 and Tables 15, 16). Diamino (2-furA-s-triazine stimulated mating activity, although no significant sterilizing action was noted. Tepa, when applied by residual film contact, at sterilizing doses, also stimulated mating activity (Table 18).
DOS E 80 pg/ male
Control
6
9
e 12
tag 40 n 15 rce e
p 18
a) 24 2
1 2 3 4 5 6
Batches of eggs
Fig. 13. Effect of trimethylmelemine on the pattern of fertility from successive batches of eggs of D. fasciatus, when males were treated by topical application, against controls.
DOSE 80 Ng/male
e---- Control U •e4 cs 3 .0
0)
.t4 a 40 - . Al. 9 co . \ I ... \ I / . \ 0 /6., 0- \ 12 a / .. - / / \ N. ../11 ■ / c / / /■ 0-- 18 / 0 0— 24
0 -
1 2 3 4 5 6
Batches of eggs
Fig. 14. Effect of pentamethylmelamine on the pattern of egg hatching of D. fasciatus when males were treated by topical application, against controls. 72
TABLE 18. Comparative sterilizing effects of 4 chemosterilants against male Dvsdercus fasciatus. All treatments were topically unless otherwise stated. (A partial summary of results given in Tables 10, 14, 15 and 16).
Dose % 21 day Eating as % Sterility *
Chemosterilant mortality of control mating lig/male
Tepa
Topical 5 0 79 100
Residual film** 15 0 117 100
Diamino (2-fury1)- s-triazine 24 0 116 6
N,N',N" tri- methyl- melamine 24 0 57 93
Pentamethyl- melamine 24 0 91 91
- Adjusted for control sterility by Abbot's formula. 2 ** lig/cm 73
DISCUSSION
Male sterility.
The data observed in Table 18,concerning the evaluation of the potential chemosterilants against male of D. fasciatus showed that tepa was the most effective sterilizing agent of those tested. Tri- and pentamethylmelamine were also quite effective, although complete sterility was not obtained with these melamines at the doses applied in the experiments.
It should also be mentioned here that although no results are quoted, formal experiments were also carried'out on the use of diamino (2-furyls-triazine. but no sterilizing activity on adult males of D. fasciatus was exhibited (Table 18).
The application of tepa caused complete sterility when applied to males either by topical or contact application, at levels which caused no observable toxic side effects. In fact the sterility index and the degree of selectivity of tepa, for males of Dysdercus, were so high, that it would be expected that this lack of toxicity and this high value for selectivity would allow treatment of males with permanent sterilizing doses without reduction of mating vigour (Tables 10 and 11).
When tepa was applied by exposing males during two hours . to a residual film, the sterility concentration (SC ) was 50 2 2.20 pg/cm ; the LC was not however determined since the 50 solubility of tepa in acetone was not enough to give an evenly high residual film on glass. It was therefore quite obvious that there was also a very wide safety margin using this method of opplicotion. 74
The s-triazines used in the present work are included in the melamine group, and they showed a very high sterilizing activity (Tables 15, 16), when applied topically to adult males, although complete sterility was not obtained in contrast with tepa.
In any sterile male work the object is to achieve 100% sterility with no loss of sexual competitiveness nor any reduction in longevity.
Thus although the SD of pentamethylmelamine was smaller 50 than the correspondent figure for trimethylmelamine, the latter s-triazine was more effective as sterilizing levels approach
100% as can be seen by comparing the slope for the regression lines of each substance (Fig. 12). This suggested that males of D. fasciatus were more susceptible to small amounts of pentamethylmelamine, but above a certain dose trimethylmelamine become more effective.
Mustafa and Yaidu (1964) had also obtained complete sterility of the red cotton bug Dislercus cinpulatus, by contact of the 2 adults with a residue of apholate at a concentration of 108.5 pecm for a period of four hours. These results showed that tepa was a much more effective chemosterilant against D. fasciatus than apholate against D. cingulatus. According to Economopoulos and
Gordon (1969) tepa was also effective against another bug,
02copeltus fasciatus, when applied topically, although this insect was more susceptible to the toxic effects of tepa than
D. fasciatus in the experiments reported here. When a dose of
40.6 pg of tepa was applied topically to adult malPs, 100% mortality was achieved 4 days after treatment and when 8.1 pg/male were 75 applied, 80.0% of mortality was obtained after 16.days, but when 206 jig/male were applied to males of D. fasciatus only
44.5% of mortality was observed after 21 days. The juvenile hormone analogue DMF also completely sterilized adults of
D. fasciatus at a dose of 5 pg/female and 50 jig/male, when applied topically (Critchley and Campion, 1971).
Recovery of fertility.
When tepa was applied, either by topical application or by residual film, some recovery of fertility was noted, although this was most marked when the second method of application was used. After topical application, recovery of fertility occurred when doses of 2.5 jig/male were applied, about 15 days after treatment, but by tarsal contact it was shown that a recovery of fertility occurred at doses of 4.0, 5.0, and 10.0 pg/cm2,
15 days after treatment in the two first concentrations and
27 days after at 10.0 jig/cm2. Males of D. fasciatus did not show any recovery of fertility after topical application of tri- and pentamethylmelamine.
Similar effects, concerning recovery of fertility, were reported by Critchley and Campion (1971) for D. fasciatus, when
DMF was applied topically at a dosage of 5 pg/female and 50 pg/male; by Economopoulos and Gordon (1969) on Oncopeltuf3 fasciatus after treatment with 10 and 20 mg/kg of tretamine; and by Gomez-Nunez
(1971) on Rhodnius prolixus, 5 weeks after treatment, when males were treated topically with doses of up to 0.10 mg of metepa per male.
The recovery of fertility ouggesta that not all the gametic cells were affected by treatment. LaChance (1969) reported that 76 if a chemical treatment produced a high level of dominant lethal mutations in the mature and nearly mature sperm, but did not succeed in killing all the gonial cells, the surviving gonial cells would continue to divide and repopulate the germarium section of the testes. Sperm derived from these surviving gonial cells would probably not contain dominant lethal mutations.
When this happened, the males might recover their fertility in later matings. Such an occurrence is more likely in long-lived insects such as the cotton stainer bug Dvsdercus fasciatus.
Recovery of fertility was also reported in other long-lived insects from Coleoptera (Reinecke et al. (1969) in Anthonomus grandis; Malik (1970) in Tribolium castaneum).
Other effects.
A study of Table 10 reveals that tepa treatment of males by topical application, did not affect the number of mating-days, the number of batches or the total number of eggs oviposited per female when applied at doses up to 2.5 fig/male, but a significant effect was observed at doses of 5.0 pg of tepa. Similar effects were shown when tepa was applied by contact but not so strongly, 2 once fecundity began to decrease at a concentration of 10 1ig/cm .
Thus application of tepa to males, topically or by residual film, affected the fecundity of females mated with them. This effect on females requires some explanation. This effect could be due to endocrine misfunction in imperfectly mated females, but the possibility of residual transfer of tepa from treated males to females during the act of mating can not be ruled out. Such a transfer would be possible since tepa aecomposition, after 77 topical application, occurred very slowly, reaching the 50% level in ELEaropsis castanea only after 45.6 h at 27°C (Campion and Lewis, 1971) whereas, as already mentioned mating took place just after treatment of the male Dysdercus with tepa.
By comparing the results obtained in both methods of application of tepa, it can be seen that there was no significant difference between the two methods with the exception of the fecundity, which was higher when residual film was used. The fecundity of all treated pairs, expressed as percentage of respective controls, was 87% for topical application and 103% for residual film. At all doses tested the fecundity was always significantly higher in residual contact treatment when compared with topical application.
Trimethylmelamine affected the numbers of mating-days, batches of eggs and fecundity per female; the differences were significant when the data were compared with those of controls.
Pentamethylmelamine had less effect.
The fact that trimethylmelamine caused a reduction in number of mating-days showed that competitiveness of the males was affected. This is not a very good recommendation for a chemosterilant. On the other hand this sterilant had an appreciable effect on fecundity at the doses tested; when females were mated with treated males they laid less than 50% of the number of eggs in the control.
It can be concluded that at sterilizing doses, tepa and trimethylmelamine affected fecundity of females mated with treated males, but pentamethylmelamine did not. Trimethylmelamine was the only one of the ehemosterilanta used that affected competitiveness of the treated males. 78
Tepa treatment of male adults affected the resulting F1 generation by reducing the number of nymphs that reached the adult stage. These effects on nymphal survival in the subsequent generation were independent of dose over the range tested (0.4 to 1.6 Fg/male).
Effect of tepa treatment on nymphs,
The results of the treatments, when tepa was applied to 3rd and early 5th instar nymphs, were significantly different from the controls; but there were no significant differences from controls when nymphs were treated as late 5th instar.
The data indicated that there was a special chemosterilant sensitivity for the early instars and for early ages of the 5th instar. Harwalker and Nair (1968) suggested that this might be explained by the fact that treatment at the late stages was not very critical for the coming moult since the epidermal cells had already been differentiated.
The dissection of males and females of the sterile pairs, at the end of the 4th week after pairing or just after death, showed that following the nymphal treatments, a majority of females were without sperm in the spermatheca though a high
percentage of males possessed motile sperm in the testes and
vesicula seminolis. These facts suggested that sperm inactivation did not take place, but that the motility of the sperm was not sufficient to achieve the transfer to the females and, therefore
the fertilization of the eggs did not occur. 79
PART TWO
THE EFFECT OF GOSSYPOL ON
DYSnERCUS FASCiATUS Signoret. SECTION A. Influence of the glandless and glanded cottonseeds
on development fecundity and fertility.
INTRODUCTIOi AND REVIEW OF LITERATURE
Occurrence of Eallual.
The poliphenolic gossypol pigments occur, as far as is known, only in the genus Gossypium, sub-tribe Hibisceae, order
Malvales (Boatner, 1948; Adams, et.al.,1960).
In 1886 Longmore isolated a crude pigment from cottonseed
oil "foots" and Marchlewski in 1899 extracted, purified, and gave
the name gossypol to this yellow pigment, the name being derived
from gossyp(ium EILLIIL to indicate its origin and chemical structure. Since then, it has been isolated from the seed and root barks and detected in other parts of the cotton plant, such
as leaves, flower buds and stems. Gossypol, as it exists in
the cottonseed, is contained in the pigment glands and constitutes
20-40 percent of their weight. They are completely formed by
the 16th day after flowering and gossypol can be detected in the
glands a few days after their formation. The content of gossypol
during development of cottonseed continues to increase until about
the 50th day after flowering, when the seed is fully matured and
bolls start to open (Caskey and Gallup, 1931). A longitudinal
section of the seed shows these pigment glands as coloured spots,
scattered throughout the tissue of the unbryo,which are relatively
large, ovoid bodies, 100 to 400 p in length (Berardi and Goldblatt,
1969) (Fir. 1 ). The colour may vary from yellow, through various
shades of orange and red, to dark purple. Increasingly dark 81
x 3.5
(a) (c)
x 3.5
(b) (d)
Fig. 1. Longitudinal sections of cottonseeds showing the pigment glands. (a) M-8 glandless (c) 247-1, high gossypol (b) M-8 glanded (d) Ashurst Lodge colours indicate the presence of gossypurpurin, another pigment, that constitutes 0.471 to 1.350 % of the weight of the pigment glands. It has generally been inferred that pigment glands are purely excretory organs in which the pigments are deposited as metabolic by products during development of the seed (Winton and
Winton, 1932; Brown, 1938).
The amount of gossypol in cottonseeds varies considerably with varieties of cotton and with the environment where it is grown (Boatner et al., 1949; Pons et al., 1953). Frampton et al. (1960) reported the total gossypol content of seeds from several species of Gossypium to range from 0.03 to 6.6'i%.
The molecular formula C30 H30 08 was proposed for gos.,ypol by Clark (1927, 1928a, 1928b, 1929). The structure 1,1'76,6',7,7' - hexahydroxy 5,5' - diisopropyl -3,3' - dimethyl (2,2' binaphtalene) 8,8' - dicarboxaldehyde, was formulated by Adams et al., 1960, (Fig.2).
The effects of dietary gossypol on animals.
In animals with a functioning rumen, dietary gossypol has not been shown to affect performance. However, symptoms of gossypol toxicity have been demonstrated when gossypol has been injected into the bloodstream (Smith, 1970). Voelcker (1858) reported, for the first time, that livestock was injured by ingestion of cottonseed meal. Withers and Carruth (1915) were the first to report the separation from cottonseedembryosof a substance which appeared to be identical with the material separated from crude cottonseed oil and named by Marchlewski, in 1899, gossypol.
They found this material to be toxic to rabbits, rats, guinea pigs and swines, and for that the toxicity of cottonseeds was entirely attributed to gossypol. S3
O H
HO OH
HO
Fig. 2. Structure of gossypol. 84
More recently, different effects of gossypol to non-ruminant animals, when they were fed on diets with various levels of gossypol, have been reported. The physiological effects of gossypol have been reviewed by Adams et al. (1960) and Eagle (1960).
Rations containing cottonseed pigment glands caused a weight depression on chickens (Groschke et al., 1947; Couch et al., 1955;
Heywang and Bird, 1955), on rats (Clark, 1927, 1928a; Gallup, 1928;
Olcott, 1948; Eagle and Bialek, 1952), on rabbits (Withers and
Brewster, 1913), and on young calves (Hollon et al., 1958).
Meanwhile, a number of investigations have shown that animals fed upon cottonseed meals with similar levels of free gossypol may show marked differences in growth and toxicity. This indicates that other factors are involved. In fact, there is some evidence showing that the level cf protein (Cabell and Eagle,
1956; Hale and Lyman, 1957; Smith et al., 1958: Clawson et al., 1961;
Sharma et al., 1966; Gallup, 1935; Cabell,. 1957; Eagle and Davies,
1957; Nairan et al., 1960; Kornegay, 1961), and quality of protein
(Eagle et al., 1956; Cabell and Earle, 1956; Cabell, 1957; Clawson and Barrick, 1957; Smith, 1957; Smith and Clawson, 1970) as well as supplement of amino-acids (Cabell and Earle, 1960) in the rations may have an influence on the toxicity of gossypol.
When cottonseed meal is incorporated in the ration of laying hens a discoloration of yolks was noticed (Heywang and Vavich, 1965;
Heywang et al., 1965). This discoloration does not appear with dietary levels of free gossypol up to 50 ppm (Smith, 1970) and can be prevented if iron in a suitable salt form were added to the chickens' ration. According to the conclusions reached at "The
Conference on Inr,ctivatior of Gossypol w-;th Mineral Sajts" if ferrous sulphate is used as a ration additive, it would be 85
possible to include significant amounts of cottonseed meal in
swine and poultry rations without undesirable effects (Anonymous, 1966) .
Withers and Brewster (1913) had already reported that rabbits
receiving iron along with cottonseed meals lived and gained weight,
but cotton meals without iron caused their death. Gallup (1928)
mentioned, also, the value of iron salts in counteracting the
toxic effect of gossypol on rats.
The effects of gossypol on insects and mites.
As mentioned previously the presence of gossypol in diets
has tremendous effects on non-ruminant animals, but the effect of
this phenolic compound on insects was first reported by Botger, et al.,
in 1964, after McMichael (1959, 1960) had demonstrated that gland-
free cottonsceds could be produced. To gain information about
the relationship betWeen the gossypol content of cotton plants and
insect resistance, many field experiments have been carried out to
compare the susceptibilities of several glandless and glanded
cotton lines to insect attack. It was observed that glandless
.lines were more susceptible to the bollworm, Heliothis zea (Boddie);
cotton leaf worm, Alabama argillacea (HUbner); beet armyworm,
Spodoptera exigua (Hubner); tobacco budworm, Heliothis virescens (F.)
(Botger et al., 1964; Maxwell, 1965; Jenkins et al., 1966;
Lukefahr et al., 1966; Wilson and lee, 1971), and to the striped
blister beetle, Epicauta vitatta (Fabricius) (Murray et al., 1965).
Maxwell et al., and Jenkins et al., in 1966, found that the boll-
weevil, Anthonomus grandis Boheman, fed significantly more on
the glanded lines, but more eggs were laid on glandless lines
(Maxwell et aid, 1966). 86
The two spotted spider mite, Tetranychus urticae Koch,
damaged the glanded-glandless pairs and high gossypol lines to
an equal extent, but the mean fecundity on the glandless pairs
was significantly greater than on its glanded parts (Schuster
et al., 1972).
To study the effect of various concentrations of gossypol
acetate incorporated into the artificial diets of larvae, feeding
tests were normally used. Such studies have been performed with
larvae of beet armyworm, LE2122Iaafaigua (Hubner); bollworm, Heliothis zea (Boddie); cabbage looper, Trichoplusia ni (Hubner);
salt-marsh caterpillar, Estigme acrea (Drury); pink bollworm,
Pectinophora gossypiella (Saunders) and _European corn borer,
Ostrinia nubilalis (Hilbner). It was observed that these
artificial diets, with various levels of gossypol, caused a
decrease in numbers of larvae, larval weight and pupal weight;
an increase in larval mortality (in some cases 100% mortality),
and in time taken for larval development and for pupal development;
and a smaller percentage of adult emergences (Botger et al., 1964;
Botger and Patana, 1966;"Lukefahr and Martin, 1966; Harding et al.,
1967; Shaver and Lukefahr, 1969).
Since the breeding of strains of cotton with very high levels
of gossypol is, in most cases, impracticable, Perkins and Canerday
(1971) carried out an experiment to determine the influence of
various concentrations of gossypol, incorporated in the diet, on
insecticide toxicity to the larvae of Heliothis zea. The results
obtained indicate that increased concentrations of gossypol
resulted in larvae being more tolerant to methyl parathion,
applied topically. 87
From this review of the bibliography it can be seen that
(1) some insect species, notably species not considered to be
pests of glanded cotton, will feed preferably on glandless cotton plants;(2) certain species, normally pests of cotton, show a
preference for glanded cotton plants;(3) some of the former species when fed on artificial diets with gossypol incorporated develop
more slowly and sometimes did not reach the adult stage. This effect is dependent of the level of gossypol in the diet.
The experiments to be described attempted to establish a relationship between cottonseed diets with different contents of gossypol, and nymphal development and reproduction ability of
D. fasciatus. The experiments to study the effect of gossypol applied topically to nymphs and adults of this species were also carried out to enlighten this very complex problem.
Determination of free gossypol.*
Several spectrophotometric methods have been proposed for the determination of free gossypol in cottonseeds (Lyman et al.,
1943; Smith et al., 1946; Hale et al., 1948). However they required extensive manipulations and employed lengthy or tedious extractions procedures or reagents lacking in specificity.
Pons and Guthrie (1949), and Pons and Hoffpauir (1955, 1957) have developed procedures based on the use of aqueous acetone as an extractant, and these have served as the basis for procedures recommended by the American Oil Chemists' Society (AOCS, 1969). This method is based in the yellow colour developed from the reaction between aniline and gossypol to form dianilinogossypol (Lyman et al.,
1943; Smith at al. 1946). The yellow colour is quickly developed by heating and is stable for at least four• hours after development.
*The term free gossypol defines gossypol vnd gossypol derivatives in cottonseed products which are soluble in aqueous acetone. 88
MATERIAL AND iET1-10DS
1. Cottonseeds.
Throughout these experiments the insects' diet have been seeds of the following cotton lines:
M-8 - glandless, free of gossypol.
M-8 - glanded, with a low content of gossypol.
247-1 glanded, with a high content of gossypol.
Ashiarst Lodge - glanded, with a normal content of gossypol. t,o4e stttas 4}144 was wwKwytogn. The first three cottonseeds were supplied by Dr. M. J. Lukefahr,
Agricultural Research Service of USDA, Brownsville, and the fourth one was ordered from British Food Manufacturing Industries, Surrey, and was so called because the name of the variety was not known.
All these cottonseeds were stored in a cool room at 5°C to reduce the possibility of changes in the gossypol content during storage.
2. Gossypol.
Gossypol acetic acid was supplied by Dr. L. A. Goldblatt,
Southern Utilization Research and Development Division of the USDA,
Beltsville, containing 89.6% gossypol by weight. It was stored into a dessicator, kept at -15°C.
3. Determination of free gossypol in cottonseeds. 3.1. ReaEents and apparatus.
a. Acetone and isopropyl alcohol, Analar grade.
b. 70% aqueous acetone (acetone-distilled water: 70-3n,v/).
c. 8o% aqueous isopropyl (isopropyl alcohol-distilled
water: 80-20, v/v). 89
d. Thiourea solution. 10 g of reagent grade thiourea
were dissolved in distilled water, diluted to 100 ml.
e. Hydrochloric acid, 1.2 N. 106 ml of concentrated
hydrochloric acid were diluted to 1 liter with
distilled water.
f. Aniline. Reagent grade aniline was distilled over a
small amount of zinc dust, and stored in the
refrigerator, and redistilled when absorbance of
reagent blank exceeded 0.022. g. Standard gossypol solution; 27.9 mg of primary
standard gossypol acetic acid were weigh ed and
transferred quantitatively to a 250 ml volumetric
flask using 100 ml of Analar acetone to effect
transfer. 1.0 ml of glacial acetic acid and 75 ml of
distilled water were added and acetone was used to
dilute to volume. From that solution.50 ml were
pipeted into a 250 ml volumetric flask, and 100 ml
of acetone and 60 ml of distilled water were added.
Acetone was added to 250 ml. As primary standard
gossypol acetic acid contains 89.6% gossypol by
weight, this standard solution contains 0.020 mg
of gossypol per ml.
h. Microid Flask Shaker.
i. Spectrophotometer Beckman, model D.B.
j. Balance Mettler H2O to weight gossypol acetic acid
and Oertling balance, model B04/15463, to weight
the samples of the cottonseed embryos, were used. 90
3.2. Calibration curve.
2, 4, 6, 8 and 10 ml aliquots of standard gossypol solution
were pipeted in duplicate, into 25 ml volumetric flasks. One set
of the aliquots was diluted to volume with isopropyl alcohol. To
the other set of aliquots 2 ml of aniline was added to develop colour.
To both sets 2 drops of 10% aqueous thiourea and 1 drop of 1.2 N HC1
were added. A reagent blank was prepared with 10 ml of aqueous
acetone, 2 drops of 10% aqueous thiourea and 2 ml of aniline. All
the volumetric flasks of the second set and the reagent blank
were heated in a boiling water bath (100°C) for 30 minutes, cooled
to room temperature and diluted to volume with aqueous isopropyl
alcohol. The corrected absorbance for each standard gossypol
aliquot was calculated using the spectrophotometer. The readings
were made at 440 nm.
A standard curve for gossypol was worked out by plotting corrected absorbances against mg of gossypol in the 25 ml volume (Fig.3).
3.3. Preparation of the samples.
10.grams of cottonseed were de-hulled and theotbryosremoved from the hulls and lint by hand screening. After they were ground in a mortar and pestle and a sample of 250 mg was used for analysis.
3.4. Gossypol determinDtion.
The samples were analysed for free gossypol by the method described in Official Methods of the American Oil Chemists' Society
(Ba 7-58, 1969). The sample was weighed in a 250 ml Erlenmeyer flask, the bottom of which was covered with a layer of solid glassbeads, and 50 ml of the 7O aqueous acetone was added. The stoppered flask was shaken for one hour on a mechanical shaker. 0.5 E e o Tr Tr m
0.3 bance r o bs A
0.1
40 SO 120 160 200 240
pg gossypol
SLandard curve for the determination of g000ypol. 92
The extract was filtered through an 11 cm circle of filter paper and the funnel covered with a watch glass to prevent evaporation.
2 ml aliquots of this filtered solution were pipeted, in duplicate, into 25 ml volumetric flasks. Further steps were equal to those used in calibration curve.
To determine mg gossypol in sample aliquots the calibration curve was used and free gossypol content was calculated as follows:
V x G free gossypol = x 100, where 1xW V = volume of the solution.
G n mg gossypol in the sample aliquot..
V = volume of sample aliquot used. 1 W = sample weight, in mg.
4. Experimental methods.
In these experiments the influence of the diet on nymphal development, and on fecundity and fertility of D. fasciatus was studied. Four different cottonseeds were used to feed cotton stainer nymphs and adults: M-8 glandless, 14-8 glanded, 247-1 and
Ashurst Lodge.
In the first experiment, to minimize differences amongst nymphs derived from different batches of eggs, four groups of 25 newly
hatched nymphs were arranged from the same batch, one for each cottonseed. Each group of nymphs was put in a plastic sandwich box (27.5 x 15 x 9 cm) with the bottom covered with damp peat to
maintain an optimum of humidity. Food and water was provided and
also a piece of plastic net to facilitate moulting. This
procedure was replicated four times.
The changes from one instar to the next, the number of
deaths and the proportions of the sexes in adult emergence were
recorded at daily intervals. 93
Nine pairs obtained from each cottonseed in the above experiment were kept, a couple in a plastic sandwich box
(19 x 11 x 8 cm), to continue the study of the effect of those cottonseed diets on the fecundity and fertility of the pairs mated. The bottom of the boxes were covered with filter paper and in each one two small plastic containers were placed: a shallow one for soaked cotton wool, to provide drinking water, and a deep one with moistened peat to provide an oviposition site for the female. On top of this peat some soaked cottonseeds were placed as food. All the following prodedures were identical to those previously described in Part One for post-treatment procedure.
5. Statistical methods. Fertility in the controls and in different treatments was compared by Chi-square test (2 x 2 contingency tables), batch by batch of eggs, and their values added to ascertain their significance, by advice of Dr. P. S. Hewlet. Means and respective standard errors were calculated using the desk computer IME 86 S.
94
RESULTS
1. Gossypol content of the cottonseeds.
In an effort to correlate gossypol content of the cottonseeds
with development, survival and ability to reproduce of D. fasciatus,
analyses for free gossypol were made of all cottonseeds used in the
experiments, before and after being eaten by Dysdercus males.
Results of these analyses are summarized in Table 1. They showed
that a definite relation existed between the species of the seed
and their content of free gossypol, but there is no significant
difference before and after feeding.
TABLE 1. Free gossypol content of different cottonseeds before and after being eaten by Dvsdercus males.
Free gossypol content % (1) Cottonseed Gossypol in Before eating After eating buds 5) (2)
M-8 Glandless gossypol-free gossypol-free 0.007
M-8 Glanded 0.98 + 0.01 MEW 0.5
247-1 3.48 + 0.01 3.52 0.01 1.7
Ashurst Lodge 1.59 0.01 1.61 .4. 0.01
(1) Calculated on the basis of weight of gossypol : weight of cottonseed embryos.
(2) Given by Dr. M. J. Lukefahr.
Comparing these results with Fig. 1 a relationship is seen
to exist between the number of pigment glands in the longitudinal
sections of the cottonseeds and their content of gossypol. 95
2. Nymphal development, fecundity and fertility of
D. fasciatus reared and maintained on glandless
and &landed cottonseeds.
The effects of the different diets were further examined by following the rates of development, and mortalities of the nymphs when reared on cottonseeds with different contents of free gossypol. - The results are summarized in Table 2.
Ashurst Lodge was the cottonseed most suitable for the nymphal development of D. fasciatus, with the smallest percentage of mortality, only 19% compared with 36, 51 and 67% in 247-1,
M-8 glanded and M-8 glandless cottonseeds, respectively. It was also noted that lack of free gossypol affected nymphal development to a greater extent than a high percentage of free gossypol (Table 2). Mortality occurred predominantly during the
3rd instar, contrary to earlier observations by Geering, in 1956 and 1960 (Fig. 4). There was no significant difference in the total nymphal development period for all of the cottonseeds used in the experiment (Table 2).
Surviving adults .of the above experiment were paired and continued to feed on the same cottonseed, as in nymphal stages, with the objective of possibly relating the ability to copulate and the capacity of reproduction with percentages of free gossypol in cottonseeds. Results of this experiment are shown in Table 3 and Fig. 5. In M-8 glandless cottonseed 33.3% of the pairs did not mate. Number of batches and fecundity were correlated with the content of free gossypol in cottonseeds and this effect was greater in 247-1 when compared with Ashurst Lodge seed. It can be seen very clearly that fertility was also affected, and was significantly higher in pairs fed on Ashurst Lodge seed •
0/0
100_ 0 0 0 > O i 0 4 0 0 O > ni 0 0 0 O rJ 0 IP!STAR o 9 0 o 0 0 O 13 C.o. 0 1st 14:4 O 0 0 O 4 O 0 > p O 71 ›:4-p-' 0 13 323 2 nd O 4 0 0 o 0'4o 0 0 50_ m 4 4• 3rd 0- O E3 > O 132 ° 41 0 O 13 > 0 O ni ><;p< • > 0 • 4th 0 O M {). O LI ().< 0 0 t> 0 0 0 O rl ;t• O 0 4>10CI • 0 0 0 0 a 5th 0 O O El > 0 0 1:1 El o • < < O El 0 0 LI 4 <1. E3 < 0 4> • < 0 a o Females > < O a O > Oro 2 4 >< 23 < 0 0 0 [rg < 0 M e 21 '10 Males
M-8 Glandless 1111-8 Glanded 2 47- 1 Ashurst Lodge
Fig. 4. Development of D. fasciatus on cottonseeds with different contents of free gossypol.
100
c Ashurst Lodge
M-8 Glanded 75
A M-8 Glandless
A 247-1
50. anwa=111111N ■••■■■■••■
1 2 3 4 5 6
Batches of eggs
Fig. 5. Influence of glandless and glanded cottonseeds on the pattern of fertility of the cotton stainer bugs, when they were fed on these varieties from the 1st instar stage. 98
than in pairs fed on M-8 glandless, M-8 glanded and 247-1 cottonseeds. The lowest fertilities were observed when pairs were fed on M-8 glandless or on 247-1 and these were significantly different from the fertilities of pairs fed on Ashurst Lodge and 1,1-8 glanded cottonseeds. There was no significant difference between pairs fed on M-8 glandless and 247-1, as far as fertility is concerned. These results suggest that both absence or high contents of free gossypol in cottonseeds affected fertility
(Table 3 and Fig. 5).
The weight of the adult cotton stainers, males and females, presented in Table 4, was also correlated with the lack or excess of free gossypol in the cottonseeds.
At the end of the experiment all males and females were dissected to examine the whole reproductive system and it was observed that testes were smaller on males fed on M-8 glandless than those fed on the other cottonseeds, and also that females fed on M-8 glandless and on 247-1 cottonseeds had abnormally small ovarioles, for the most part with occytes only in the apical third part. This effect was greater on 247-1 (6 in
9 females) than on M-8 glandless (2 in 6 females), and explains the low fecundity observed in females fed on these cottonseeds
(Tables 3 and 4). 99
TABLE 2. Dysdercus fbsciatus development on cottonseeds containing different contents of free gossypol.
No. surviving adults Cottonseed No. 1st Nymphal instar period nymphs Males Females Total Males: (days) (a) females
M-8 Glandless 100 15 18 33(a) 0.83 27 (22-30)
M-8 Glanded 100 22 27 49(b) 0.81 27 (22-32)
247-1 100 26 38 64(c) 0.68 27 (23-30)
Ashurst 100 48 33 81(d) 1.45 26 (23-28) Lodge
(a) Numbers followed by the same letter are not significantly different at the 5% level of probability.
TABLE 3. Fecundity and fertility of adult females of D. fasciatus reared and maintained on cottonseeds with different contents of free gossypol
(a) Cottonseed No. pairs Mating Number No. eggs "aerkfemale Fertility Formed Mated -days batches Laid Hatched
M-8 Glandless 9 6 9.3 5.7 663+24(a) 523+37 78.9(a)
M-8 Glanded 9 9 10.3 6.5 704+36(a) 634+67 90.1(b) 247-1 9 9 14.3 4.8 523+70(b) 397+88 75.9(a)
Ashurst 9 9 15.0 7.3 897486(c) 842+83 95.o(c) Lodge
(a) Percentages followed by the same letter are not significantl;,.- different at the 1% lovel of probability. 100
TABLE 4. Effect of various concentrations of free gossypol in cottonseeds on the weight of adults, on the testes size, and on the ovaries.
Cottonseed Mean weight (mg), Testes size (mm) Females at 4 weeks (1) with.fully developed Males Females Length Width ovarioles (%)
M-8 Glandless 74.3 244.6 1.57 0.72 66.6
52.0(2) 188.0(2) (1.45-1.65) (0.61-0.87) m-8 Glanded 90.6 288.6 1.68 0.72 100.0 (1.57-1.88) (0.66-0.78) 247-1 76,4 234.0 1,72 0.76 33.4 (1.59-1.38) (0.72-0.87)
Ashurst Lodge 84.o 279.1 1.87 0.78 100.0 (1.77-1.97) (0.64-0.87)
(1) Average of nine pairs; in M-8 glandless only six pairs. (2) Average weight of adults that did not mate. 101
3. Reproduction rate of adults fed on different cottonseeds
after emergence, in relation to their content of free
gossypol.
Table 5 shows the results obtained when pairs of cotton stainers were allowed to feed on cottonseeds with different contents of free gossypol during the whole of their adult life.
Competitiveness of the males was not affected since the number of mating-days was not significantly different from each other, but the number of batches and consequently the number of eggs laid per female were smaller in 247-1 cottonseed, suggesting that a high content of free gossypol had a detrimental effect on fecundity.
Fertility was also affected and was significantly higher in the pairs maintained on Ashurst Lodge than in the pairs maintained on M-8 glandless, M-8 glanded and 247-1 cottonseeds; it was also significantly lower in pairs maintained on 11-8 glandless than in pairs on Ashurst Lodge, M-8 glanded and 247-1 cottonseeds
(Table 5, Fig. 6).
The lowest percentage of eggs hatching was observed in batches from pairs fed on M-8 glandless, suggesting the possibility that some gossypol may be necessary to produce high fertility.
4. Comparative index
Bearing in mind that the initial immigration into the cotton crop by adult stainers starts in February - March (Pearson, 1958;
Sweeney, 1q60; Anonymous, 1968), permitting them to breed a second generation in the crop, and the results obtained from the experiments in which D. fa7;ciatus was reared and maintained on cottonseeds with different contents of free gossypol, a comparative Voss0024. index was worked out to indicate theXtrends in an insect population 102
TABLE 5. Reproduction rate of adults fed throughout adult life on cottonseeds with different contents of free gossypol.
Cottonseed No. Mating No. No. eggs per female Fertility pairs -days batches % (a) Laid Hatched
m-8 Glandless 9 17 6.7 744+75 543+53 72.9(a)
M-8 Glanded 9 23 7.0 802+48 660+56 82.3(b)
247-1 9 20 6.2 684+76 555+73 81.1(b)
Ashurst 9 18 7.1 784+50 707+51 90.2(c) Lodge
(a) Percentages followed by the same letter are not significantly different at the 1% level of probability. 100
et) cy a C '75 • Ashurst Lodge 8)
o M-3 gianded
A 247-1
▪ M-3 glandless
50
1 2 3 4 5 6 7 8
Batches of eggs
Fig. 6. Influence of glandless and glanded cottonseeds on the fertility of egg batches from cotton stainers fed on these varieties. lc* bred on those cottonseeds, without any other factor exerting harmful effects on the populations concerned, and considering that the four cotton fields were invaded by the same number of couples.
These indices are given in Table 6. They were calculated on the assumption that Ashurst Lodge cottonseed is the most suitable to breed D. fasciatus.
The cumulative comparative indices obtained show that in just one season the cotton stainer bug population built up on
Ashurst Lodge cottonseed (the most suitable for cotton stainer development and reproduction, in the conditions of these experiments) was about 13, 5 and 4 times greater than the cotton bug populations built up on M-8 glandless, 247-1 and M-8 glanded, respectively.
105
.144.1141. TABLE 6. Fecundity and fertility perAfemale, and number of resulting adults from adult pairs fed on different cottonseeds. Indices for the 1st and 2nd generations, and also the final cumulative comparative index, were expressed as percentage of the relevant figures for D. fasciatus when they were fed on Ashurst Lodge cottonseed.
1st. Generation 2nd. Generation Compara- Cottonseed tive Eggs Eggs Result- I * Eggs Eggs Result- I .3 index laid hatched ing 1st. laid hatched ing 2nd. (Cumula- adults adults tive effects) m-8 Glandless 744 543 179 31 663 523 172 25 7.7 m-8 Glanded 802 660 323 56 704 634 311 46 25.8 247-1 684 55o 355 61 523 397 254 37 22.0
Ashurst Lodge 784 707 573 loo 897 842 662 loo 100.0
* Comparative index for the 1st and 2nd generations, occurring in the 'same cotton season. 106
DISCUSSION
Little has been written concerning the influence of different food materials on development time and reproduction among seed- feeding insects.. The literature mostly concerns the nutritional requirements of the insects, based on the influence of chemically defined diets on development. By rearing nymphs on cottonseeds with different contents of gossypol, the suitability of the different seed strains as food was studied and some information obtained on their effects on development and reproduction of the cotton stainer bugs.
The determinations of free'gossypol in the cottonseeds used in these experiments showed that appreciable differences existed between them. Although the percentages of gossypol vary, for the same variety, with the environment where the cotton plants were grown (Pons et al., 1953). Such differences within strains are likely to be small compared with the differences between strains.
The results of the analyses of free gossypol in cottonseeds, before and after being eaten, showed that there is no preference for the pigment glands in relation to the other parts of the e mbryo The analyses also showed that a relationship could be drawn between gossypol content of the cottonseeds and flower buds (Table 1).
In the present studies the cotton stainer nymphs could be reared to adult stage on all diets tested, but there were differences in the survival rates. These differences can probably be ascribed to the amount of free gossypol present in the cottonseeds. The highest mortality during nymphal development was found on M-8 glandless and most deaths occurred during the third instar or 107
during the moult to the fourth instar. Survival was poor on
M-8 glanded; many nymphs persisted into the fifth instar and some of them died before starting to moult or during the final ecdysis. The nymphal mortality was not so high on cottonseed
247-1, but the number of surviving adults on all of these cottonseeds was significantly less than on Ashurst Lodge, a normal glanded cottonseed, which was therefore considered the most favourable for nymphal development of D. fasciatus. These results suggested that free gossypol is required for nymphal development, up to a certain percentage, considered to be an optimum value, after which harmful effects were again apparent.
The development time of nymphs reared on these different cottonseeds was not significantly different. The sex ratio
was affected; more male than female nymphs died on the unsuitable cottonseeds. Males:females ratios were ranging between 0.83 to o.68 for Dysdercus fed on m-8 glandless, M-8 glanded and 247-1
cottonseeds, and was equal to 1.45 for cotton stainer bugs reared
on Ashurst Lodge cottonseed (Table 2).
Parallel effects, correlated with gossypol content of the
cottonseeds, were observed in relation to fecundity and viability
of the eggs of the surviving adults from the experiment mentioned
above. Effects on fecundity were more marked in adults fed on
high gossypol than on low gossypol cottonseeds, but fertility was,
practically, the same. Both, fecundity and fertility were significantly lower when the insects were reared on any other
than the optimal Ashurst Lodge seed.
The effects of the various diets on fecundity and viability
of the eggs were less accentuated when insects started to feed
on them just after they became adults, after feeding during the 108
nymphal life on normal glanded cottonseeds. It is known that food obtained as a nymph may have a great effect on the reproductive capacity of certain insects (e.g. Wigglesworth, 1960; Johansson, 1964).
Fertility was significantly lower on M-8 glandless, 14-8 glanded and 247-1 cottonseeds, but the effect was most marked in the case of the M-8 glandless seed. In this case, a larger difference was observed in the last three egg batches, possibly because all
the gossypol ingested during nymphal life has already been absorbed or excreted by that time (Figs. 5 and 6).
Thus, all the results obtained suggested that there was a correlation between the effects described above and the absence or excess of free gossypol in the diet provided to nymphs and adults. Previous authors have shown that the seeds of Malvales VW:AA s Lit.COPI t lc" are occential to development of Dysdercus and they have made some suggestions to explain the results obtained (Rainey, 1948; Geering and Coaker, 1960; jiodjat, 1963). The results obtained in these experiments, relating content of free gossypol of the cottonseed diets, nymphal development and reproduction of D. fasciatus, would explain satisfactorily those results.
Rainey (1948) suggested that a specific protein might be required for successful breeding, admittedly with a different species (D. nigrofasciatus), and that this protein only occurred in the Malvales. It could equally well be, however, that the essential requirement is a fat-soluble factor common to the
Malvales which increases in the developing bolls as suggested by Geering and Cooker (1960). With the knowledgement of the results obtained in these present experiments it seems very likely that the "protein" reported by Rainey, the "fat soluble factor" mentioned by Geering and Coaker, and gossypol are one and 109
the same chemical substance, a suggestion which would reconcile the results reported by those authors.
This hypothesis is supported by a better development of nymphs on older bolls (Pearson, 1934 and 1937) and by comparing the statement made by Geering and Coaker (1960):
"The results obtained from the experiments in which bolls
of different ages were fed to the adult stainers may
possibly indicate that factors necessary for oviposition
increase in quantity as the age of the bolls increases.
A change in concentration of these factors would produce
the observed differences in rate of egg-production" with the fact that content of gossypol increases with the age of the bolls, being maximum when seed is fully matured (Caskey and Gallup, 1931), and also with the results reported by Hodjat
(1963). D. fasciatus fed with boiled cottonseed diet laid fewer eggs than those in normal food. Possibly the explanation for that result is based on the fact that boiled cottonseeds have less gossypol than normal cottonseeds, because gossypol combines, under the influence of heat, with either the free amino acids or free carboxyl groups of cottonseed protein to form bound (1) gossypol , that is inactive (Boatner, 1948).
Another fact, also mentioned by Geering and Coaker (1960), is that stored cottonseed may be less suitable for egg production than freshly harvested cottonseed. In fact, Podolskava, cited by
(1) Bound gossypol is defined as the portion not extracted directly by aqueous acetone, and is calculated as the difference between the values obtained for total and free gossypol.
110
Castillon et al., 1949, found that during storage of variety 114
of Gossypium hirsutum the content of gossypol decreased from 1.15
to 0.755 during storage for four months. This result provides a possible explanation for the data obtained by Geering and
Coaker in the effect of storage of seeds on the fecundity of
Dysdercus. On the other hand Boatner et al., 1949, reported
that in the 10 varieties investigated, gossypol was not found to follow a consistent pattern of change, when stored for several months at temperature of 27 + 3°C.
It is likely that the different contents of free gossypol in
the diets of the nymphs resulted in adults that were lighter when they were fed on M-8 glandless or on high gossypol 247-1 than on M-8 glanded or Ashurst Lodge cottonseeds. Absence or a high content of gossypol in cottonseeds led to the same results, suggesting that the insects show a marked preference for cottonseed of optimal gossypol content, but feeding preferences should be discussed later (Table 4).
The comparative indices that were calculated for the Dysdercus
populations fed on the cottonseeds used in the experiments, using as a reference the most suitable cottonseed (Ashurst Lodge), showed that the cotton stainer population reared on M-8 glandless would: increase more slowly than those ones reared on 247-1 and
M-8 glanded. The increase in these populations was about 13, 5 and 4 times smaller than on the population reared on Ashurst Lodge cottonseed, respectively (Table 6). This information may be of importance in any programme to control this pest. These effects
can be considered as a result of antibiosis, a term that was
proposed by Painter (1968) for those adverse effects on the insect life history which result when the insect uses a resistant host-
plant species or variety for food. 111
SECTION B. 12122Ekst2smssoa toicalionnnronsand
adults.
INTRODUCTION
A survey of literature concerned with the influence of gossypol on nymphal development and potential of reproduction of insects shows that most work has been based on effects of the artificial diets in which gossypol was incorporated, or on field experiments designed to reveal the extent of antibiosis due to glandless and glanded cotton plants, with the one following exception, reported by Botger et al.,(1964). When nymphs and adults of Aphis gossypii and adults of 20L1=1119._EmIlisltlurbeElpe were caged on cotton plants sprayed with acetic acid solution containing 5% gossypol,
100% mortality of the aphids resulted in 24 hours, but only 40% mortality of the thurberia weevills was observed, after four days.
As D. fasciatus cannot be reared and maintained on artificial diets, gossypol was administered to nymphs and adults, by topical application, to determine its effect on development and reproduction of this cotton pest.
Toxic effects were also studied by applying gossypol topically on males or by injecting it into the cottonseeds on which they were fed. 112
MATERIAL AND PETHODS
1. Topical application of gossypol on nymphs and adults.
After finding the range of effectiveness of gossypol in prelithinary experiments, the dose of 24 pg was chosen for treating nymphs and doses of 1.2 and 12.0 pg were selected for applidation to Dysdercus males. Immediately before the application, a solution of gossypol was prepared by dissolving 40 mg gossypol acetic acid in 1 ml Analar acetone, from which dilutions were made when necessary. 0.6 111 of the prepared solutions were applied topically on 50 2-4 days 5th instar nymphs, and on nine males 0-2 days after moulting, per dose.
Treated nymphs were observed daily to record deaths or any abnormality until they moulted to adults. Ten pairs of adults were then withdrawn from controls and from treated individuals and each pair was placed in a plastic sandwich box (19 x 11 x 8 cm) and maintained on the same diet.
All males, treated as adults with different doses of gossypol, were paired with virgin females of the similar age and the pairs were put in the usual plastic sandwich boxes. Controls consisting of males treated only with acetone were observed, also.
Post-treatment procedure was the same as previously described in Part One.
2. Evaluation of toxiciti
A solution containing 160 mg gossypol acetic acid in 2 ml
Analar acetone was prepared just before use.
In the topical application 2 x 1 pi containing 20 to 80 pg of gossypol were applied on 50 adult males, 0-2 days after 113 moulting. When gossypol was injected into the 247-1 cottonseeds,
20 p1 of the solution prepared for the toxicity tests containing
1.6 mg gossypol, were used. Two groups of 20 adult males, each group starved for three days before the experiment, were allowed to feed on those cottonseeds during a period of four days. A third group of 30 adult males, as control, was fed on 247-1 cottonseed injected only with 20 p1 of acetone. After that all of them were fed with untreated 247-1 cottonseed.
The responses of D,ysdercus males to the topical application and to the gossypol injected cottonseeds were recorded daily for a period of three weeks.
The solutions of gossypol were applied topically to the insects with a Burkhard micro-drop applicator fitted with a one ml
Agla-all-glass syringe and 20 G needle. To inject gossypol into the cottonseeds a 100 pl Terumo micro syringe (NSN - 100 Shandon) was used. RESULTS
1. Topical application on 5th instar nymphs.
Results of the topical application of gossypol in acetone
to 5th instar nymphs, 2 - 4 days after moulting, are shown in
Tables 7 and 8. There was no significant difference in the number becoming adult when treated nymphs are compared with controls,
but the number of deaths occurring up to 10 days after moulting
to the adult state was significantly larger in the group of adults resulting from treated nymphs than in the control group
(P <0.001). The capability of reproduction of mated pairs, originating from treated nymphs,.was greatly affected; a decrease in number of mating-days, batches of eggs and eggs laid per female was observed 1,41en compared with controls. The fertility of treated insects was also significantly less than the fertility of controls (P <0.001) (Table 8).
2. Topical application on males.
Results of the combination of the gossypol applied topically to Dysdercus males and feeding on cottonseeds with different contents of free gossypol are presented in Table 9. Contrary to expectation the effect of gossypol applied topically was found to depend on the gossypol content of the cottonseed on which the cotton stainer bugs had fed. Thus, in insects fed on M-8 glandless or N-8 glanded (without gossypol or with a low percentage, respectively) the topical application of 1.2 pg of gossypol, in acetone, per male, increased the fertility significantly. The increase in fertility Was more marked for insects fed on glandless seeds (Table and Figs. 7 and 8). 1.3.5
TABLE 7. Mean % mortality of D. fasciatus adults when 5th instar nymphs were treated with 24 ig gossypol acetic acid.
Dose No. nymphs Resulting adults Deaths Fg/nymph treated Males Females Total % 10 days after % * moulting 142 24 50 19 26 45 90.0 5 d'e 42.2(a)
CONTROL 50 23 20 43 86.6 0.0(b)
TABLE 8. Reproductive ability of resulting adults from 5th instar nymphs treated with 24 Fs gossypol acetic acid.
Dose No. Mating- No. No. eggs No. eggs Fertility Fg/nymph pairs days batches laid hatched % *
24 10 6.5 6.0 735 503 64.3(a)
CONTROL 10 12.5 8.5 1023 782 75.4(b)
* Percentages followed by the same letter are not significantly different at the 1% level of probability. 116
TABLE 9. Effect of gossypol acetic acid, applied topically, on fecundity and fertility of D. fasciatus feeding on cottonseeds with different contents of free gossypol.
Cottonseed No. 'gating- No. No. eggs per female Fertility days batches o, and pairs * dose(Fg/male) Laid Hatched
M-8 Glandless
Control 9 17 6.7 744 + 75 543 + 53 72.9(a)
1.2 9 20 7.3 769 + 37 655 + 48 85.2(d)
12.0 9 20 6.2 702 + 97 540 ± 97 76.9(a)
M-8 Glanded
Control 9 23 7.0 802 + 48 660 + 56 62.3(b)
1.2 9 23 7.9 847 F 47 749 + 42 88.4(d)
12.0 9 20 6.8 750 + 66 654 + 77 87.2(d)
247-1
Control 9 20 6.2 684 + 76 555 + 73 81.1(b)
1.2 9 20 6.1 674 + 89 540 + 77 80.1(b)
12.0 9 20 7.3 767 + 43 632 ± 43 84.0(b)
Ashurst Lodge Control 9 18 7.1 734 + 50 707 + 51 90.2(c) 1.2 9 18 6.9 751 + 71 610 + 64 81.2(b) 12.0 9 24 7.3 84o + 71 671 + 53 79.9(b)
* Percentages followed by the same letter are not significantly different at the 5% level of probability. 117
When the same dose was applied to males feeding on cottonseeds with a normal content of gossypol (Ashurst Lodge variety), it was observed that the effect was precisely the opposite, since fertility was significantly less in treated insects than in controls (Table 9 and Fig. 9). There was no effect at all on insects fed on 247-1 cottonseed, with its very high content of gossypol (Table 9, Fig. 10).
It is interesting to note that the effects resulting from the higher doses applied were not significantly different from the lower for all cottonseed diets except for M-8 glandless. This fact may
possibly be explained, for the former cottonseeds by suggesting that the higher dose exceeds the optimum, but when insects fed on glandless seeds probably the absence of gossypol and the higher dose were equally displaced on either side of the optimum value, causing low fertility in both cases (Table 9, Fig.11).
The results for females paired with male adults treated with
12 pg of gossypol and fed on Ashurst Lodge cottonseed suggested
there was a tendency to increase the number of mating-days and fecundity, but analysis showed these increases were not significant.
Pairs receiving the same treatment and fed on 247_1 seed appeared
to show the same tendency in respect to fecundity but again the
differences were not significant. It is a feature of the present
work that for reasons beyond the author's control the number of
pairs in the successive experiments were low and it is possible
that further work with larger number of insects might well disclose
significant differences in the fecundity of the females fed on
these cottonseeds. 100
CI
*"(:), . ,0 0 . ./ S. P 75 .. . i-, . / co Co 'ID Control C 0 ai 1.2pg/male
50
1 2 3 4 5 6 7 8
Batches of eggs
Fig. 7. Effect of gossypol, applied topically, on fertility of D. fasciatus feeding on M-8 glandless cottonseed. 100
------ca '0------0
as 75 Control C.
1.2 tag/male EtZ
50
1 2 3 4 5 6 7 8 Batches of eggs
Fig. 8. Effect of gossypcl, applied topically, on fertility of D. fasciatus feeding on M-8 glanded cottonseed. a Cont rol
0 1.2 .g/male po 0
1 2 3 4 5 6 7 8
Batches of eggs
Fig. 9. Effect of gossypol, applied topically, on fertility of D. fasciatus feeding on Ashurst Lodge cottonseed. 13 Control
/ I ■ 0 1.2 lug/ male
2 3 4 5 6 7 8
Batches of eggs
Fig. 10. Effect of gossyrol, applied topically, on fertility of D. fasciatus, feeding on 247-1 high gossypol cottonseed.
1001
••■• - e --- 0.• tag n e erc p A PA-8 Glandless
C A Glanded
0 247-1
o Ashurst Lodge
Control 1.2 12
DOSE (pg/rnale )
Fig. 11. Influence of doses of gosoypol, applied topically, on fertility of D. fasciatus, when they were fed on cottonseeds with different contents of free gossypol. 123
3. Mortality effects
The results from the experiments conducted to determine whether gossypol in itself is actually toxic to adult males of
D. fasciatus are presented in Table 10, when gossypol was applied topically, and in Table 11, when gossypol was injected into the cottonseeds on which males were allowed to feed afterwards.
In both experiments the mortality observed, within three weeks, was not significantly different from controls, even when
160 ug of gossypol was applied topically to males. Thus gossypol is not toxic to male Dysdercus in spite of huge doses applied.
Comparing the mortality effects of gossypol when applied on adult
males and on 5th instar nymphs it was observed that much smaller
doses applied to nymphs disturbed the following ecdysis causing
a high proportion of abnormal adults that died prematurely.
More females died than did males (7% females and 25% males
(Tables 7 and 10).
124
TABLE 10. Effect of various doses of gossypol acetic acid on mortality of D. fasciatus males.
Dose No. males treated Mortality at 3 weeks pg/male
o/ No. males dead /0
4o 5o 1 2.0 So 5o 2 4.o 16o 5o 5 10.0
CONTROL 60 2 3.3
TABLE 11. Mean % mortality of D. fasciatus males after feeding on 247-1 cottonseeds in which gossypol, in acetone, was injected.
Treatment No. males Mortality at 3 weeks
No. males dead /c/
Cottonseeds injected with 1.6 mg gossypol 40 6 14.7 (a)
CONTROL. 30 0 0.0 (a)
Percentages followed by the same letter are not significantly different at the 5%. level of probability. 125
DISCUSSION
When gossypol was applied topically to 5th instar nymphs, the dose applied (24 pg/nymph), did not cause the death of the nymphs, in numbers significantly different from the controls but resulted in the early appearance of many dead adults. In fact, the number of deaths observed, within 10 days after adult emergence, was significantly greater than in controls. Fecundity and fertility of the surviving adults from treated nymphs, were significantly less than in controls. Thus, it can be concluded that application of gossypol to 5th instar nymphs affected both, development and the reproductive capacity of the D. fasciatus.
Gossypol applied topically to adult males, 0 - 2 days after moulting, had varying results on fertility, depending on the content of free gossypol of the cottonseed on which they had been maintained after becoming adults. On cottonseeds with no or with a low percentage of free gossypol, the smaller dose applied increased fertility significantly but the higher dose did not affect fertility when compared with controls. On cottonseeds with high gossypol content the application of gossypol did not have any effect on the egg viability, but on Ashurst Lodge, a normal glanded cottonseed, both high and low doses caused a significant decrease in fertility.
These results (showing that doses of gossypol as small as
1.2 pg may have significant effects on adult males) are difficult to reconcile with the fact that the insects may consume much larger quantities of gossypol, by feeding on glanded seed, without apparent effect.
The experiments reported in the next Section were designed to gain more information which might help to explain these results. 126
In toxicity tests, either when very high doses of gossypol
were applied topically or when gossypol was administered to them
by injecting 1600 pg in each cottonseed, no correlation was found,
between dosage and mortality of the males, indicating that gossypol is not toxic to this cotton pest in the adult stage. 127
SECTION C. Uptake and excretion of goss7221.
INTRODUCTION
The results obtained in previous experiments indicate that
when cotton stainer bugs were reared and maintained on certain
strains of cottonseed, development, fecundity and fertility were
better and higher than when bugs were reared on others. Another important factor, is that topical application of the small amounts
of gossypol, such as 1.2 lag per male, had a great effect, mainly
on fertility of fernales paired with treated males.
On the other hand, O. W. Richards discussing a paper presented by Johansson (1964) to the "Symposia of the Royal Entomological
Society of London : Number Two", on Insect Reproduction, pointed out that many nutrition experiments are badly designed, because it is not known how much food is ingested.
Because of these factors, it was considered advisable and interesting to correlate all the results obtained with the amount of food consumed by males, to know how much gossypol was taken in, to determine how much free gossypol there is in frass and in the gut, and also the comparative preferences of the insect for glandless and glanded cottonseeds.
Experiments were carried out in order to obtain relevant data on these various aspects of the uptake and excretion of gossypol. 128
MATERIAL AND METHODS
1. Conversion factor.
A factor to convert dry weight to normal weight was determined for M-8 glandless, 247-1 and Ashurst Lodge cottonseeds.
Similar quantities of these three seeds held a constant humidity were weighed before being soaked for about 12 hrs. After that they were placed in the constant temperature room for 48 hrs and then dried in an oven at 100°C for two days. The dry weight was then determined.
The conversion factor (F) was calculated as follows:
Normal weight F - Dry weight
2. Amount of food eaten.
To determine the amount of food eaten per insect and per day,
Ashurst Lodge cottonseed was offered to Dysdercus. Males, recently becoming adults, were placed in plastic boxes (27.5 x 15 x 9 cm),
20 in each, and they were allowed to eat in the same soaked cottonseed for a period of 48 hrs. These seeds were weighed before soaking (A) and their dry weight was determined after eating (B). The amount of cottonseed eaten per insect and per day was then found by the following formula:
g of food eaten = A - BF 2n where F is the conversion factor and n the number of insects used in the experiment. 129
3. 221-nparative preference.
Three different cottonseeds were used in these studies:
M-8 glandless, 247-1 and Ashurst Lodge. Twenty males were selected just after becoming adults and placed in plastic boxes
(27.5 x 15 x 9 cm), with the bottoms covered with filter paper,
where they had a free choice between M-8 glandless and Ashurst
Lodge or between M-8 glandless and 247-1. Soaked cotton wool
was used to provide drinking water to the insects. Cottonseeds
were weighed before being soaked in water. After having been
eaten by the cotton stainers for a period of 48 hrs., their dry
weight was determined by the usual method. The amount of food
consumed by each male, per day, was calculated by the above
formula. These procedures were repeated four times for each
strain of cottonseed.
4. Free gossypol in the frass.
Ten male cotton stainers, immediately after becoming adults,
were placed in plastic sandwich boxes (19 x 11 x 8 cm), the bottoms
of which were covered with filter paper to collect the frass.
This filter paper was changed every second day and stored in a
deep freezer prior to analysis.
Thin layer chrometocraphy. The method used was basically that
of Bell (1967), but with some small alterations introduced by
Dr. G. N. J. le Patourel. Frass was extracted from the filter
paper with ethanol (four extractions each of 5 ml) and transferred
to a 100 ml -enaratoring funnel to which Pn ml of distilled water
i:as added. The resulting soli tons i. r e extracted three_ times
with 50, 20 and 20 ml of ether. The ether was collected in
100 ml conical flask, dried over anhydrous sodium sulphate, 130 transferred to a 100 ml pear flask and reduced to dryness.
Residues were dissolved in benzene and applied to thin-layer chromatography plates (TLC).' For comparison a solution of pure gossypol in benzene was applied on the same line of the TLC plates.
This solution of pure gossypol was prepared by allowing an ethereal solution of gossypol acetic acid to evaporate over water and collecting the crystals of gossypol, that were dissolved in benzene. Two recrystallizations were made to obtain pure gossypol.
Frass used for the spectra and chromatograms were from males fed on Ashurst Lodge (Figs. 12, 15 and 16) and on M-8 glandless cottonseeds (Figs. 17 and 18).
Chromatograms were developed with 95% benzene - 5% methanol
(Fig. 12) and"50% toluene - 40% ethyl formate - 10% formic acid
(Figs.14,16 and18). Phenolic compounds were characterized by spraying plates with 1% phloroglucinol in 2NHC1.
Free gossypol analysis. The filter paper was cut into small pieces which were put in 250 ml volumetric flasks and the determination of free gossypol was done as described previously.
These determinations were carried out on frass from Ex_s12ELRE males after feeding on Ashurst Lodge cottonseed throughout all their lives or after feeding on that cottonseed during nymphal stages and on M-8 glandless after emergence. Each determination was replicated four times.
All these experiments were conducted in a constant temperature room maintained at 27 + 0.5°C and 70 + 5% relative humidity. 131
5. Free gossypol in the gut.
Forty Dysdercus males, four days old, were dissected. The guts were removed and placed in an Erlenmeyer flask with 50 ml of aqueous acetone and the bottom covered with glassbeads. Each determination was replicated four times and 10 guts were used in each one. The method previously described to determine free gossypol was followed.
Dissections were performed with alive insects pinned on their backs to a black paraffin dissecting plate, after wings and legs have been removed.
6. Statistical methods.
Student's t-distribution was used when necessary. Means and standard errors were calculated with the desk computer IME 86s. 132
RESULTS
1. Amount of food consumed.
To find out how much cottonseed was eaten by a standard number of insects in a standard time it was necessary to determine a factor to convert dry weight in cottonseed normal weight. The relevant data is presented in Table 12.
TABLE 12. Determination of a factor to convert dry weight in normal weight of the M-8 glandless, 247-1 high gossypol and Ashurst Lodge cottonseeds.
Cottonseed Replicate Normal weight Dry weight Conversion Seed index (g) (g) factor - F (g)
I 11.092 9.823 1.129 NI-8 10.362 9.172 1.129 9.97 + 0.13 Glandless III 10.448 9.201 1.136
Mean and standard - - - 1.132 error 0.002
1 10.835 9.464 1.145 247-1 II 10.963 9.621 1.140 12.68 + 0.29 III 10.728 9.369 1.145
Mean and standard 1.143 + error 0.002
I 10.987 9,638 1.140
Ashurst II 10.771 9.414 1.144 10.07 + 0.15 Lod.g..e III 11..230 9.845 1.141
Mean and standard 1.1 42 + error 0.001 133
As it can be seen the conversion factors of the three cottonseeds
were very similar in spite of the existing differences of their seed
indices (weight of 100 seeds).
The amount of Ashurst Lodge cottonseed consumed by batches
of Dysdercus males was determined in order to find the mean quantity
of gossypol ingested daily, by each male. The data obtained are
shown in Tables 13 and 14, and illustrated in Fig. 19.
The analysis for free gossypol (Table 1) indicated that
Ashurst Lodge cottonseed contains 1.59%, and Table 14 indicated
that each male consumed 5.67 mg of that cottonseed embryo
per day. Thus it was possible to estimate the amount of free
gossypol ingested per male and per day (90 jig).
2. Free gossypol in the frass
After an abundance of gossypol had been demonstrated
qualitatively in the frass of Dysdercus males fed on Ashurst Lodge cottonseed containing 1.59% free gossypol (Figs. 12, 15 and 16),
a more accurate knowledge of how much gossypol there is in the frass was required. For that reason, analyses of free gossypol in the frass were carried out and the results obtained are given in Table 15. In Fig. 19 the lines for cumulative values of free gossypol ingested and recovered in the frass were plotted over a
period of six days immediately after emergence, showing that 45.4%
of the free gossypol ingested was excreted through the frass during
that period of time.
134
TABLE 13. Quantity of Ashurst Lodge cottonseed ingested by 20 Dysdercus males, every 48 hrs, during the first six days after adult emergence.
Replicate Normal weight Dry weight Converted weight Quantity ingested. (g) (g) (g) (g)
13.055 11.244 12.836 0.219
I 13.062 11.232 12.822 0.239
13.070 11.311 12.912 0.157
12.970 11.168 12.749 0.221
II 13.003 11.221 12.810 0.194
12.984 11.199 12.784 0.200
12.970 11.125 12.699 0.271
III 12.993 11.245 12.837 o.156
13.251 11.427 13.044 0.207
13.093 11.274 12.870 0.223
IV 13.021 11.054 12.618 0.402
12.152 10.430 11.907 0.246
13.012 11.179 12.761 0.251
V 13.025 11.302 12.902 0.124
13.008 11.139 12.716 0.292 135
TABLE 14. Quantity of Ashurst Lodge cottonseed ingested per male and per day.
Quantity ingested Replicate (mg) (20 males each) 0 - 2 days 2- 4 days 4- 6 days TOTAL
I 219.0 239.0 157.0 615.0
II 221.0 194.0 200.0 615.0
III 271.0 156.0 207.0 634.0
IV 223.0 402.0 246.0 871.0
V 251.0 124.0 292.0 667.0
Total per 100 males 1185.0 1115.0 1102.0 3402.0
Mean quantity and s.e. per 5.57 4. 1.32 5.51 + 0.56 5.67 ± 0.40 insect and 5.92 + 0.26 per day (mg). 136
TABLE 15. Quantity of free gossypol in the frass of Dysdercus males when they were fed on Ashurst Lodge cottonseed throughout their life.
Replicate Free gossypol (pg/male/day) 0 - 2 days 2 - 4 days 4 - 6 days TOTAL
I 65.00 20.00 27.50 112.50 II 65.00 20.00 30.00 115.00 III 55.00 27.50 27.50 110.00 IV 52.50 36.25 33.75 122.50
TOTAL 237.50 103.75 118.75 460.00
Mean and standard 59.38 + 3.29 25.94 + 3.86 29.68 + 1.48 115.00 + 2.70 error 137
TABLE 16. Quantity of free gossypol in the frass of Dysdercus males, when they were fed on Ashurst Lodge cottonseed during the nymphal period and on M-8 glandless, after adult emergence.
Replicate Free gossypol (ug/male/day)
0 - 2 days 2 - 4 days 4 - 6 days Total
I 46.25 1.25 2.50 50.00
II 53.75 3.75 2.50 6o.00 III 61.25 2.50 7.50 71.25
IV 55.00 15.00 10.00 80.00
TOTAL 216.25 22.50 22.50 261.25
Mean and standard 54.06 4- 3.08 5.62....4.3.17 5.62 4- 1.87 65.30 4 6.54 error 133
Fig.12. Thin layer chromatogram of frass extracts of D. fasciatus, when males were reared and maintained on Ashurst Lodge cottonseed (a), and pure gonsypol (b). Developing solvent: 95r benzene - methanol: stationary phase: silica gel (0.5 mm); spray: 1;', phloroglucinol in 2N hydrochloric acid.
139
2 E ANC 1 SORB AB
0
280 380 480
WAVE LENGTH ( nm)
Fig. 13. SpectruL of pure gossypol.
Pig. 14. Thin lover chromaLogram of pure gobsypol. Fig. 15.Spectrumofgossypolandderivativesinfrass. Fig. 16. Thin layerchromatogram ofgossypol andderivativesin frass (a),and puregossypol (b).
AB S O RBANCE 280
WAVE LENGTH(nm) b 1.40
380 a
b 480 11+1
2 ANCE 1 RB SO B A
0
280 380 480
WAVE LENGTH (nm)
Fig. 17. Spectrur: of frass extract (no gossypol in frass).
b a
Fig. 18. Thin layer chromatogram of frass extract (no gossypol) (a), pure gossypol (b). 1 1, ^) .A. TC
600
Ingested 500 CD 0 A Excreted E L. 0 400
0
a)
200 E
100
Ira IMII•■••■■•• •■■••■••■11
1 2 3 4 5 6
Days after adult erner9ence
Fig. 19. GossypoI ingested and excreted by Dysdercus males when feeding on Ashurst Lodge cottonseeds. 1.
100
1
1 2 3 4 5 6
Days after adult emergence
Fig. 20. Excretion of gossypol by Dysdercus males just after becoming adults. Nymphs were fed on a glanded cottonseed (Ashurst Lode) and tnale adults on N-8 glandless cottonseed. 144
3. Free aampl in the gut
It had already been demonstrated that free gossypol was
excreted in the frass, and it was also shown that gossypol continue
to be excreted on the sixth day after adult emergence, even if they
were fed on glandless cottonseeds since that date, indicating
that free gossypol consumed during nymphal life remains in the
gut for some time (Table 17 and Fig. 20).
To determine the quantity of free gossypol retained in the
gut of Dysdercus males, analyses of the gut were performed when
males were 4 days after moulting, and the results are presented in Table 17.
4. The comparative preference of D. fasciatus for glandless
and glanded cottonseeds.
The results of the experiments in which cotton bugs had a
choice of two different cottonseeds, in. respect of gossypol content, are shown in Tables 18, 19 and Fig. 21. When the choice was
between a cottonseed with an optimum content of gossypol and another one without gossypol, there was a significant preference for the first one (t = 3.07), but there was no significant
difference in preference from one or another cottonseed, when the
choice was between a cottonseed with a high content of gossypol and a glandles3 cottonseed (Tables 13, 19). The total a:lount
of food consumed in the first experiment was 41% greater than in
the second experiment, mainly because the cotton stainers showed
a marked preference for Ashurst Lodge cottonseed, with a content
of free gossypol that may be considered to be the optimum, from
the results obtained in the experiments previously described. 145
TABLE 17. Amount of free gossypol in the gut of Dysdercus males, on the 4th-day after adult emergence.
Replicate Free gossypol 1g/male
I 60.0 II 95.o III 85.o IV 70.0
Mean and standard error 77.5 + 7.7
146
TABLE 18. The comparative preference of Dysdercus fasciatus males for M-8 glandless and Ashurst Lodge glanded cottonseeds.
(A) Food consumed by 20 males in 48 hrs.
Cottonseed Replicate Normal weight Dry weight Converted weight quantity (g) (g) (g) ingested (mg)
I 11.101 9.783 11.068 •33.o M-8 II 11.181 9.840 11.133 48.8 Glandless III 10.163 8.927 10.100 63.2 IV 11.069 9.731 11.009 59.8
I 10.342 8.782 10.025 316.9 Ashurst II 11.200 9.643 11.008 191.1 Lodge III 11.169 9.672 11.041 127.3 IV 11.256 9.750 11.130 126.3
(B) Food consumed from glandless and glanded cottonseeds per male in 24 hrs.
Cottonseed Food consumed Food consumed .20 males/2 days male/day /mg (mg)
M-8 Glandless 204.8 1.28 + 0.17 (a)
Ashurst Lodge 761.6 4.76 + 1.12 (b)
Total 966.4 6.04 + 0.95
02 Numbers followed by different letters are significantly different at the 5% level of probability. 147
TABLE 19. The comparative preference of Dysdercus fasciatus males for M-8 glandless and 247-1 cottonseeds. (A) Food consumed by 20 males in 48 hrs.
Cottonseed Replicate Normal weight Dry weight Converted weight quantity (g) (g) (g) ingested (mg)
I 10.115 '8.855 10.018 96.9 m-8 II 10.289 9.029 10.215 74.6 Glandless III 10.765 9,459 10.702 63.5 iv 10.825 9.474 10.718 107.0
I 10.666 9.227 10.547 118.7 247-1 II 10.716 9.316 10.649 66.6 III 10.361 9.051 10.346 15.3 iv 11.141 9.672 10.999 141.4
(B) Food consumed from glandless and high gossypol cottonseeds per male in 24 hrs.
Cottonseed Total food consumed Food consumed (mg) male/day (mg)
M-8 glandless 342.0 2.14 4- 0.25
247-1 342.0 2.14 0.70
Total 684.0 4.28 + 0.95 Fig. 21.Cumulativevaluesoffood ingested whenDysdercusadultmales
Cumu lative v a lues of food E 300 200 500 700 400 100 600 have acho3c glandless andhigh gossypolcottoned.
0-2
,, betweenrlandle- 2-4 1 1
+8 DAYS , -= andnormr.!1rlanded orbr.twoen 4-6
G-8 o • • 1111-8 Maudless reil-8Gland;ess Ashurst Lodge 247-1
149
DISCUSSION
The comparative preference.
The relative preferences of the male cotton stainer bugs for glandless, normal glanded and high gossypol cottonseeds were studied by giving different batches choices of two cottonseeds: glandless or normal glanded, and glandless or high gossypol.
In the first experiment there was a marked and significant
preference for normal glanded against glandless cottonseed, indicated by the amount of food consumed by the males from each cottonseed. Stanford and Vilhover (1918) reported that pigment glands secrete a volatile oil that may be responsible for the characteristic odour of glanded cotton plants. Assuming that
this suggestion is correct, it is possible that the difference in food consumption observed in the experiment may be attributed
to the attraction exerted by that odour. Once cotton stainers arrived and stayed on cottonseeds that attracted them, the feeding response might be regulated in part by odour and taste given by
the presence and percentage of gossypol in cottonseeds; though some feeding occurs in the absence of gossypol.
In the second experiment, where the choice lay between
glandless and high gossypol cottonseeds, there was no difference in the amount of food infested by males from each seed. Perhaps,
as neither seed is the ideal food for them, as can be seen by
comparing the total amount of food consumed in each of theSe
experiments (Tables 18 and 19), they ate from both, thus balancing
their diet. 150
Gossyyol in the frass.
The quantities of cottonseed ingested were estimated by the difference between weights before and after eating, using a factor to convert dry weight of the seeds to normal weight.
In the experiments individual feeding rates were not considered but only rates of groups of 20 males from which, means were calculated.
When provided with Ashurst Lodge seed, each male consumed
5.67 mg of food a day, from which it may be estimated that the intake of free gossypol was about 90 lig per day.
By thin layer chromatography it was shown that gossypol was excreted in the frass and that amount was determined by calorimetric methods (Table 15). The free gossypol excreted in six days after adult emergence represented only 45.5% of the free gossypol ingested per male, but the excretion of gossypol takes quite a long time, because six days after being changed to a diet without gossypol, males continue to excrete gossypol consumed during nymphal stages (Table 16 and Fig. 19). 151
GENERAL DISCUSSION
The present investigations demonstrated the effects of chemosterilants, glandless and glanded cottonseeds, and gossypol applied topically to the males on the reproductive potential of the cotton stainer bug Dysdercus fasciatus Signoret. The results have been' discussed in appropriate sections of this work. Some of the aspects of this study, however, not discussed earlier and their implications, mainly concerning the control of this species, are discussed here.
Breeding methods.
The results obtained in some of the experiments with chemosterilants had to be rejected because of the high natural sterility observed in the controls (30 - 50%), therefore these experiments were repeated. Other workers using this insect have encountered similar problems with the fertility of their cultures (personal communication : B. Critchley and C. Wall).
One hypothesis advanced for such a high percentage of natural sterility was that this effect might be due to residual insecticides applied in the fields or fungicides applied as seed-dressing treatment after.delintage. Neither of these suggestions seemed very probable, the first because, generally, the application of insecticides (e.g. carbaryl, DDT) is stopped when the bollS start to open and the period of time for the cottonseed to reach this country is so long that all insecticide residues might be expected to be degraded. The second one is even more unlikely since no cottonseed for the oil industry receives any treatment, unless a mistake had occurred, and in this case the contamination would already have be,r>n detected by the o-; 1 ginneries. 152
As a result, the last experiments with s-triazines were
carried out with insects from a new culture, built up from some
nymphs obtained from Reading University, and in July 1972, a
third culture was built up from some adults imported from Malawi.
In the new cultures only low levels of natural sterility were
observed ( 5n. In all experiments carried out in Part two
of this research work, and concerned with the influence of
gossypol on D. fasciatus, the nymphs and adults used were from
'Malawi strain.
Since, in some experiments using Malawi strain, no sterile
pairs were observed in the control treatments. Therefore it
can be concluded that the natural sterility occurring previously
was due to inbreeding, since that original strain had been
reared in Silwood Park insectary, at least since 1967 (personal
communication of Mr. Ives, in-charge of the Dysdercus cultures,
at Silwood Park).
This same phenomenon was reported by MacGill (1942)
comparing a number of generations of Dysdercus howardi Ballou,
bred in laboratory between 1932 and 1934, with a similar number
of generations bred from 1940 to 1941. In the later generations
there was a higher percentage of infertile eggs; the difference
between the two groups of insects being highly significant.
To minimize the possibility of the effect recurring, the
breeding methods were changed to those previously described (p.14 )
with successful results. A sub-culture from this population
has been independently reared over a number of generations without
encountering any fertility problems (Miss Sheila Balnave : personal
communication). 153
Fecunditz, fertility and gossypol
It is known that differences in quantity as well as in quality of food consumed by the larvae may result in differences in fecundity. Moreover, in insects which lay eggs in batches, as happens in D. fasciatus, renewed food ingestion by adults is often required before the next batch is produced. Thus, there maybe a connection between food ingested and fecundity
(Johansson, 1964).
The results obtained from the experiments using cottonseeds with different contents of gossypol indicated that the presence of gossypol, up to an optimal value, may be necessary to make food palatable and attractive to the cotton stainers, since they consumed much more food from glanded than from glandless and high gossypol cottonseeds (Tables 18 and 19).
These differences in food consumption resulted in malnourished adults with reduced weight, and reduced numbers
of ovarioles in females (Table 4). Such reductions may be the
cause of the low fecundity observed when compared with adults fed on normal glanded cottonseeds (Tables 3 and 5). These
effects were more marked when D. fasciatus was reared and maintained
on the same seeds than when they were reared on a glanded cottonseed
and the adults were maintained on seeds with different contents
of gossypol (compare Tables 3 and 5).
The effect of the presence and proportions of gossypol in
the amount of food ingested was also observed by iaxwell et al., 1966.
In feeding tests made with various dilutions of pure gossypol it
was found that 0.1 - 1.0concentr3tion of the compound in agar
plugs produced a feeding response in Anthonomus grandis,
indicating that the presence of gossypol enhances feeding. 154
It would be very interesting to know at what concentration
gossypol begins to inhibit feeding of the boll weevil.
The absence of gossypol may be the reason for the reduction
of the viability of the eggs from females feeding throughout
their lives on glandless cottonseedS (Table 3), because the
omission of special substances may have a profound influence
on egg viability. Sterols, for instance, seem to play an
important part in the fertility of Muses domestics, although
the omission of sterols from the diet has little effect on
ovarian growth or total egg production (Johansson, 1964). It
is necessary to emphasize again that a percentage of gossypol
in the cottonseeds above the optimum was also correlated with
reduction in fertility (Table 3).
Topical application of gossypol
The reasons why a small dose of gossypol, such as 1.2 pg,
applied topically to the males, had so great infuence on the
viability of the eggs, are thought to be:
Much of the ingested oss ,pol is excreted in the frass. In
. fact, about half of the consumed free gossypol had been excreted
by the end of the experiment. If the amount existing in the
gut on the fourth day after adult emergence (2.6 times more than
the amount excreted on the same day) is added to the amount excreted
until that day, this total represents about -?; of total free
gossypol consumed.
Considering the amount of gossypol ingested and excreted,
the simplest hypothesis is to conclude that the difference between
these two values represents gossypol absorbed by insect. But this
would. represent doses of the order of 45 - 50 itg per day. 155
In spite of that yet applied doses of only 1.2 pg of
gossypol had significant effects. Therefore the results suggest
the conclusion that most of the ingested gossypol, which is not
excreted, must be metabolised either in gut lumen or as it is
absorbed through the gut. cells. But it is also possible that after being absorbed may combine with proteins to form bound gossypol that is physiologically inactive.
These conclusions require confirmation and would form the basis-for some further experiments of interest. A study to know how much gossypol there is in the body of the insect, and the amounts of gossypol existing in the gut and excreted in the frass throughout their whole life (nymphs and adults) might help to understand this phenomenon.
General nutrition is known to have profound effects on reproduction in insects. According to Wigglesworth (1960) and
Johansson (1964), whereas females reproduction may be strongly influenced by nutrition, male reproductive activity is little affected. On the other hand, Johansson (1958) reported that starvation does not produce infertile males in Oncopeltus, but the testes and vesicula seminales are somewhat smaller than those of fed males of the same age. The results of the present experiments are similar to the latter results since the size of testes were smaller in males fed on M-8 glanuless and 247-1 cottonseeds when these were compared with males bred on Ashurst
Lodge, the most suitable cottonseed (Table 4). The results strongly suggest this is probably a direct effect of reduced feeding (Tables 18 and 19). But the fact that gossypol applied 156
to such males increases the fertility of pairs suggests it
may have in addition a more direct physiological action, compensating for the reduced size of testes observed in males fed on cottonseeds with a content of gossypol below the optimum.
On the other hand, when insects were maintained on cottonseeds with a gossypol content about the apparent optimum, the results show that topical application of administered gossypol caused a decrease in fertility. This could be due to excessive levels of gossypol having an effect on spermatogenesis or causing dominant lethal mutations or affecting sperm transfer.
What is clear is that the fertility decreases in gossypol treated insects fed on normal cottonseeds to the level observed in insects maintained on high gossypol cottonseeds. In the latter, the fertility was always the same, either in controls or treated pairs; it is suggested that this is because the amount of gossypol in the seed and ingested by such females was already enough to depress the fertility to the level observed.
The fact that the highest dose applied (12.0 lig per male) did not have any effect in fertility of the pairs maintained on
M-8 glanded, 247-1 and Ashurst Lodge cottonseeds, compared with the small dose (1.2 ug per male), suggests that above a certain dose, that is different for each cottonseed, there is no increase in the effect of gossypol applied topically in every strain; higher gossypol treatments were correlated with low fertilities.
It may be helpful to note that anyone planning further work on the inhibition of reproduction in Dysdercus fasciatus should be aware that, firstly the fertility and fecundity of the laboratory culture should be carefully checked (for the species seems very susceptible to degenerating in culture over a period of time) and 15'7
secondly that the long generation time is a limiting factor in the rate at which experiments may proceed. Nevertheless some interesting problems remain to be investigated. These include the following:
1. The fate of that fraction of the ingested gossypol
(about 50.%) which is not excreted. Is gossypol o, ()MU«, ht. 0464,At& partially metabolisedXin the gut as it slowly passes
through? How much is absorbed through the gut wall
into the tissues, and how rapidly is it metabolised
in the tissues?
2. The effects of gossypol injected directly into male
Dysdercus, notably upon sperm fertility and transfer.
The development of the ovaries and spermatogenesis
when nymphs and adults are fed with cottonseeds with
different contents of gossypol.
Dysdercus control.
From the results obtained with tepa treatments on males of the cotton stainer bug D. fasciatus it can be seen that it is poSsible to sterilize males at very low doses, when the sterilant is applied either topically or by residual film. Tepa and other alkylating agents produce mutagenic effects, and hence would be too dangerous to apply directly in the field. But such compounds would perhaps be used under closely controlled conditions for sterilizing mass-reared or mass-collected insects for subsequent release. 158
The experiments with s-triazines showed also that certain
melamines have pronounced sterilizing effects on male of D. fasciatus.
The fact that some s-triazines (e.g. simazine, ipazine and atrazine)
have been used in the field as herbicides raises a possibility that some may be used in a method of direct application or more
probably in combination with an attractant or a bait-station.
This species was originally controlled very efficiently, before
the appearance of the synthetic organic insecticides, with bait- sprays, consisting of a sweetened solution of sodium arsenite
which was applied to the cotton crop at the time of the first immigration of Dysdercus (Pearson, 1958). This same approach could perhaps be followed using the most promising s-triazine, trimethylmelamine, instead of sodium arsenite.
On the c'ther hand, the information gained from the experiments with glandless and glanded cottonseed could be very useful for breeding resistant plants to this species. In fact, the
potential decrease in the rate of growth of cotton stainer
population reared and maintained on glandless or even on high gossypol cottonseeds. indicated by these experiments was so great that this aspect should not be overlooked in any integrated control programme for the whole cotton pest complex.
Although very high and glandless seeds both restrict Dysdercus multiplication, in practice more interest attaches to glandless or low gossypol seed strains because these are more valued by the cottonseed oil industry.
However, it is necessary to emphasize that each insect species has a specific reaction and that therefore when considering the entire range of species feeding on cotton plants, no general
conclusion that glandless cotton varieties are more insect- X59
resistant or more insect-susceptible should be drawn. All these facts indicate the need for entomological testing of glandless and glanded lines to determine their effects on specific insect pests. Meanwhile, as gossypol is harmful to some other cotton pests that attack leaves, flower buds and flowers (notably lepidoptera, e.g. Heliothis zea and Alabama arFillacea), perhaps the best cultural answer to these various
pests having different reactions could be given by plant breeders if they could manipulate levels of seed and floral gossypol separately to produce low gossypol levels in cottonseed and high gossypol level in floral parts, as was suggested by Wilson and
Lee, (1971). Under these conditions information about the relationship between seed and floral gossypol content would be
particularly important (Table 1).
Finally, there is the problem of when glandless cottonseed
can be grown commercially?
Harper (1962) reported that he "can see nothing which suggests
that commercial production in the one-variety area of California
will be delayed beyond 1970; however, this is not a certainty
by any means". In 1970, Hosfield et al. reported that glandless
lines were still yielding from 5 to 10% less fibre than their
recurrent parents, depending upon the location of the test, and
in 1972, Professor R. F. Smith said, that the problem remained,
since a glandless cotton variety has not yet been obtained with
the same agronomic properties of the glanded ones (personal
communication). Way The work described in this thesisXprovidet an additional
reason for encouraging the development of commercial glandless
cotton varieties for it has been shown that cotton fields of 16o
C.4. )act, such varieties ohould be less heavily infested with Dysdercus and thus less insecticide may need to be used in relation to a given yield. 161
SUNYARY
PART ONE
1. The structure of the reproductive system of Dysdercus fasciatus Signoret, and the alignment and the functions of the different parts during copulation were studied. The existence of a structure considered to be an erection fluid pump is reported for the first time.
2. Tepa was applied topically to adult males of D. fasciatus and complete sterility was obtained with 5.0 pg/male. When adult males of this cotton stainer bug were treated by exposing them to a residual deposit of tepa similar results were obtained with a 2 concentration of 15.0 pg/cm . Sterility doses (SD50) were very low : 0.88 pg/male for topical application and 2,20 pg/cm2 for residual contact.
3. The sterility index (LD50/SD50) and the degree of selectivity (LD50 /SD90') were very high, 2591. and 56, respectively.
4. A recovery of fertility occurred approximately 15 days after treatment at a dose of 2.5 pg/male, after topical application, 2 and at concentrations of 4.0 and5.0 pg/cm , after residual 2 exposures. At a concentration of 10.0 yg/cm , recovery occurred
27 days after treatment.
.1• The number of mating-days and batches of eggs, fecundity, fertility and pattern of hatchability during the experiments were determined for both methods of application. ‘dith the exception of fertility, theae properLies were noL affected by topical 162 application of tepa at a dosage of up to 2.5 Fg/male, but were affected when doses of 5.0 jig/male were applied. All figures, others than fertility, were very similar to those of the controls when tepa was taken up from residual deposit; concentrations of
10.0 and 15.0 ig/cm2 reduced fecundity. (At the levels tested, the number of batches and fecundity per female were higher when tepa was applied as a residual film than When it was applied by topical application).
6. Treatment of 3rd instar nymphs with 10 and 15 ig of tepa per nymph, by topical application, reduced the number of subsequent adults. Similar effects were obtained when tepa was administered to early 5th instar nymphs, but the treatment had no effect when late 5th instar nymphs were treated (5 to 50 lig per nymph).
7. The reproductive potential of the adults resulting from treated 3rd and early 5th instar nymphs was affected. The number of mating-days and batches of eggs for adults derived from treated
3rd instar, and the number of batches of eggs only, for the adults derived from treated early 5th instar, were smaller than in the controls. The majority of dissected females of the sterile pairs had no sperm in the spermatheca, but most of the males had motile sperm in the testes and vesicula seminalis. This indicated that sterility, in such circumstances may be due to a disturbance in the mechanisms of sperm transfer.
8. Tepa treatment of the male Dysdercus affected the development of the nymphs in tile F1 tenerotion; the number of resulting adults was significantly lower than in controls. 163
9. Tri- and pentamethylmelamine induced 93.2 and 91.2% sterility, respectively, when doses of 24 jig/male were topically applied to adult males D. fasciatus, but the same dose of diamino (2-fury1)-s-triazine did not induce any sterility.
10. Although sterility of all treated insects was not obtained at doses applied, there was no recovery of fertility in those which were sterilized.
11. Trirnethylmelamine was more effective than pentamethyl- melamine when SD values were compared (24 and 73 Female, 95 respectively).
12. At these levels trimethylmelamine affected the number of mating-days, egg-batches and fecundity, but pentamethylmelamine did not.
PART TWO
13: Free gossypol contents of the N-8 glandless, 14-8 glanded,
Ashurst Lodge and 247-1 high gossypol cottonseeds were determined and a definite relation was found between varieties of seed and those contents.
14. Results obtained from feeding tests with those cottonseeds showed that the Ashurst Lodge variety was the most suitable seed for nymphs and adults of D. fasciatus; its content of gossypol is 1.59, based. on weight of cottonseed embryos. 164
15. The number of resulting adults was significantly reduced
when Dysdercus nymphs were fed on cottonseeds with no gossypol
(M-8 glandless) and with small (M-8 glanded) or high content of gossypol (247-1), when compared with Ashurst Lodge cottonseed.
16. The sex ratio (male/female) was greatly reduced when nymphs were reared on cottonseeds with a gossypol content below or above the optimum (0.83 (M-8 glandless): 0.81 (M-8 glanded) and 0.68 (247-1) compared with the control ratio of 1.45); i.e. males were more susceptible to seeds providing dietary or phagostimulant deficiencies.
17. The number of mating-days and batches of eggs, fecundity and fertility were reduced when D. fasciatus was reared and maintained on cottonseeds with reduced or high content of gossypol.
18. Absence or excess of gossypol in the diet cause abnormal ovaries in which oocyte development is partially inhibited, resulting in the low fecundity, mentioned above. This effect was more marked in females on 247-1 high gossypol cottonseed, when 66.6% of females
had abnormal ovaries.
19. D. fasciatus adults reared and maintained on i-8 glandless and 247-1 high gossypol weighed less than those on Ashurst Lodge cottonseed.
20. Fecundity and fertility were also reduced when adults were
fed on the latter cottonseeds. The results, although significantly
different from those obtained using rishurst Lodge variety, did not show
the same marked differences as those exhibited by insects reared and
maintained on these cottonseeds throughout the entire life cycle. 165
21. From results obtained in the above experiments the
rates of multiplication of Dysdercus reared throughout over
two generations (corresponding to one cotton season) on the
different strains of cottonseed were deduced and compared.
Assuming the same number of pairs initially, after two generations
the populations of Dysdercus reared on Ashurst Lodge variety would
exceed those on M-8 glandless, M-8 glanded and 247-1 varieties by factors of 13, 4 and 5, respectively.
22. Topical application of 24 pg of gossypol on 5th instar nymphs, 2 - 4 days after moulting, had no effect on the number of resulting adults, but provoked earlier deaths of these adults when compared with controls.
23. Numbers of mating-days were reduced by about 50 and fecundity and fertility were significantly less in adults resulting from the above treated 5th instar nymphs compared with controls.
24. When topical applications of gossypol were applied to
Dysdercus males the effects on fertility varied with the content of free gossypol in the cottonseeds on which they had fed. When the cottonseeds'- content of free gossypol was below the•optimum, the topical application of gossypol improved fertility significantly; when the content of free gossypol was near the optimum the application of gossypol reduced significantly the fertility, but when content of free gossypol was above the optimum, there was no effect in fertility of the pairs formed. 166
25. Gossypol applied topically on Dysdercus adult males at
very high doses (160 pg per male) did not increase mortality.
Other batches of males fed on 247-1 high gossypol cottonseeds which
has been injected with gossypol (1.6 mg per seed) did not exhibit
a significant increase in mortality; these insects had been
starved for three days prior to feeding, fed for four days on
gossypol injected seeds, then maintained for three weeks on
247-1 cottonseed.
26. The amount of food consumed by twenty males in two days
was determined in order to estimate the intake of free gossypol
per male and per day.
27. Free gossypol in the frass was determined qualitatively
by thin layer chromatography and quantitatively by colorimetric
methods. Free gossypol in the gut was also determined.
28. When nymphs were reared on Ashurst Lodge and the resulting adult males were maintained on M-8 glandless cottonseeds, gossypol ingested during the nymphal life continued to appear in the frass for six days after adult emergence.
29. Dysdercus males exhibited a marked and significant
preference for glanded cottonseed (Ashurst Lodge), when a choice between this and M-8 glandless seed was Given to them. When
the choice was between M-8 glandless and 247-1 high gossypol,
Dysdercus males showed no preference. The total amount of food consumed when M-8 cinndiess and 747-1 seed were offored represented only 7Oi of the total food consumed in the first choice, when
ALihurst Lodge seed was offered with giandieso seed. 167
30. It is concluded that some but not all the effects that have been correlated with abnormal levels of gossypol may be correlated with the reduced food intake. 168
ACKNCWLEDGENENTS
This dissertation is dedicated to my loving wife for her patience, tolerance and encouragement during the course of this work; and to my sons for their understanding of all the implications
of these three long years.
I am extremely grateful to my supervisor Dr. C. T. Lewis for
his constant help and advice but especially for his astute criticism of the manuscript.
I am also indebted to Dr. D. G. Campion, my supervisor during
the first part of this investigation concerning chemosterilants, for his helpful suggestions, encouragement, and also for his criticism of the draft of Fart One.
I wish to acknowledge Dr. G. N. J. le Patourel for his helpful assistance with chemical analyses, and for reading and discussing the draft of Part Two.
I should also like to thank Dr. A. B. Borkovec for providing s-triazines; Dr. L. A. Goldblatt for the samples of gossypol acetic acid; and Dr. N. J. Lukefahr for the glandless and glanded cottonseeds.
I am also grateful to Professor T. R. E. Southwood for providing working space and facilities at the Field station; and to
Dr. B. E. Natthews for the scanting electron micrograph;
R. H. Williams for the photograr,hy; and V.r. J. Ellis for
his help in moinLaining the constant temperature room. 169
Finally, my special thanks go to the Calouste Gulbenkian
Foundation for granting the scholarship that made these studies possible and to the University of Lourenco Marques for their assistance. 170
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