STUDIES ON THE COPULATORY BEHAVIOUR OF THE FREE-LIVING
NEMATODE PANAGRELLUS REDIVIVUS (GOODEY, 1945).
by
C.L. DUGGAL
M.Sc. (Hons. School) Panjab University
A thesis submitted for the degree
of Doctor of Philosophy in the
University of London
Imperial College Field Station,
Ashurst Lodge,
Sunninghill,
Ascot, Berkshire. September 1977 2
AtSTRACT
The copulatory behaviour of Panagrellus redivivus is described in detail and an attempt is made to relate copulation with the age and reproductive state of the nematodes.
Male P. redivivus show both pre- and post-insemination coiling around the female and they use their spicules for probing and for opening the female gonopore. Morphological studies on the spicules have been made at both the light microscope level and the scanning electron microscope level in order to understand their functional importance during copulation. The process of insemination has been studied in some detail and the morphological changes occurring in the sperm during their migration from the seminal vesicle to the seminal receptacle have been recorded. It was found that during migration the sperm formed long chains by attaching themselves anterio-posteriorly, each sperm producing pseudopodial-like projections.
The frequency of copulation in the male nematodes and its influence on the number of sperm produced and on the nematode life- span was examined, and compared with the development and longevity of aging virgin males. The number of sperm shed into the uterus of the female at the time of copulation was found to increase with increasing intervals between copulations.
Similar observations were also made on the life-span and oocyte production in copulated and virgin females. The phenomenon 3
of sex attraction was studied in relation to oocyte development in
the female's reproductive tracts and was investigated using 4th stage
larval females, and virgin and non-virgin females of varying age
and reproductive states. Virgin females were found to be attractive
to and respondent towards the adult males whereas copulated females neither attracted nor responded to adult males.
The timing of the first copulation of the newly moulted female was studied in relation to the number of available males.
The possible effects of age on the first copulation of a virgin female was also noted. Further copulation in inseminated females was studied in relation to storage of the oocytes and the number of sperm present from the previous copulation. It was found that large numbers of sperm can delay further copulation considerably but that aged sperm in the seminal receptacle did not prevent further copulation. 4
ACKNOWLEDGEMENTS
My thanks are due primarily to my supervisors,
Professor N.A. Croll for suggesting the problem; Dr. A.A.F. Evans for his continual interest and advice; and Dr. D.J. Wright for his constant help, advice, and encouragement throughout this work.
My appreciations are due to Professor T.R.E. Southwood for providing work space and facilities at the Field Station.
My thanks are also due to Mrs. A.J. Budd for typing this manuscript and all those at Imperial College Field Station who have helped me in so many ways.
Finally, my special thanks go to the Government of India for providing me with a scholarship during the tenure of this work. CONTENTS
Page
Abstract 2
Acknowledgements 4
Contents 5
Introduction 10
General copulatory behaviour and physiology in nematodes 10
Sex attraction in nematodes 16
Female copulatory activity in nematodes 18
Males copulatory activity in nematodes 22
Sex ratio in nematodes 25
Conclusions 27
Section I - The experimental animal and its culture 30
in the laboratory
I.A. Panagrellus redivivus (Goodey, T., 1945), 30
Order Rhabditida (Oerley, 1880), Family
Panagrelaimidae (Thorne, 1937)
I.B. Monoxenic culture 30
I.C. Preparation of agar plates 31
I.D. Observation on P. redivivus at low magnification 32
I.E. Observation on P. redivivus at high magnification 32
I.F. Scanning electron microscopy 34
1.Tail region of adult male P. redivivus 34
2. Sperm 34
Section II - Observations on the general reproductive 36
biology of P. redivivus
II.A. The male reproductive system 36 6
Page
II.B. Male accessory reproductive organs 40
1.The spicules 40
2. The gubernaculum 40
3. Caudal papillae 43
II.C. The female reproductive system 43
II.D. General copulatory behaviour of P. redivivus 45
1. Pre-insemination coiling of the male 45
2. Penetration 49
3. Insemination 49
4. Insemination in the post-vulvar uterine sac 51
5. Post-insemination coiling of the male and its 52
attachment to the female
II.E. Maturation of sperm in the female 53
1.Introduction 53
2.Experimental procedure 55
3. Observations 56
Section III - Copulatory behaviour of male P. redivivus 66
III.A. Post-moulting copulatory behaviour of adult 66
male P. redivivus
1.Experimental procedure 66
2. Results 66
III.B. The rate of copulation, the number of sperm 68
transferred and the life span of normally
copulating males
1.Experimental procedure 68
2. Results 68 7
Page
III.C. Development and life span of virgin male 71
1.Experimental procedure 71
2. Results 72
III.D. Effect of short isolation periods on the 73
copulatory behaviour of•male P. redivivus
1.Experimental procedure 73
2. Results 73
2a. The duration of pre-insemination coiling 76
2b. The number of sperm transferred per copulation 76
2c.Duration of post-insemination coiling 79
and attachment
III.E. Effect of long isolation periods on the 80
libido of male P. redivivus
1.Experimental procedure 80
2. Results- 81
Section IV - Development and life span in female P. redivivus 83
IV.A.1. Introduction 83
2. Experimental procedure 83
3.Results 84
3a.Development in virgin females 84
3b.Development in copulated female 92
3c.Mortality of virgin and copulated females 93
Section V - Sex attraction in P. redivivus 95
V.A. Introduction 95
V.B. Test apparatus for sex attraction experiments 95
and experimental procedure 8
Page
V.C. Results 98
1.Response of virgin females and adult males 98
towards E. coli
2. Homosexual attraction and response of virgin 100
females and adult males
3. Attraction and response of fourth-stage larval 100
females to adult males
4. Attraction and response of normally copulating 102
females to adult males
5. Attraction and response of aging virgin females 102
to adult males P. redivivus
Section VI - Copulatory behaviour of females P. redivivus 107
VI.A. Time of first copulation in female 107
1.Introduction 107
2. Experimental procedure 107
3. Results 108
VI.B. The effect of aging on the first copulation 110
of the virgin female
1.Introduction 110
2. Experimental procedure 110
3.Results 111
VI.C. Factors influencing the second copulation 111
1. Introduction 111
2. Experimental procedure 114
2a. The influence of male copulatory activity on 114
the copulatory behaviour of once-copulated females 9
Page
2b.Number and aging of sperm in the uterus 114
2c. Number and aging of oocytes in the female 115
3. Results 115
3a.The influence of male copulatory activity 115
on the copulatory behaviour of once-copulated
females
3b.The number and aging of sperm in the uterus 117
3c.The number and aging of oocytes in the females 121
VI.D. The third copulation of female P. redivivus 124
1. Experimental procedure 124
2. Results 124
VI.E. Behaviour of sperm of P. redivivus in vitro in the 126
presence of oocytes
1.Introduction 126
2. Experimental procedure 126
3.Results 126
General Discussion 127
Copulatory behaviour and the role of the spicules and 127
caudal papillae during copulation
Functional maturation of sperm in nematodes 129
Copulatory behaviour of male nematodes 133
Sexual development of adult female nematodes 135
Sex attraction in nematodes 138
Copulatory behaviour of female nematodes 142
Conclusions 146
References 149
Appendices 158 10
INTRODUCTIO N
The reproductive physiology of nematodes has recently
been reviewed by Anya (1976) and Lee and Atkinson (1976). It is
apparent that little is known about the factors influencing
copulation in nematodes and that the process of copulation itself has been described in relatively few species.
General copulatory behaviour and physiology in nematodes
In most of the nematode species examined the male coiled its tail around the vulval region of the female using its specialised copulatory muscles to form a loop through which the female moved forwards and backwards several times prior to copulation (Greet, 1964; Chin and
Taylor, 1969; Trudgill, 1976). In a few species, however, the male appeared to move the ventral surface of its cloacal region backwards and forwards over the cuticular surface of the female until it located the vulval opening (Fisher, 1972; Chuang, 1962; Anderson and Darling,
1964; Jones, 1966; Sommerville and Weinstein, 1964; Somers, Shorey and Gaston, 1977). At copulation the anterior end of the male and female Nematospiroides dubius were found to point in opposite directions
(Sommerville and Weinstein, 1964), whereas in Cylindrocorpus spp. they were at right angles (Chin and Taylor, 1969). During copulation the head of the male and female of Ditylenchus destructor were never seen to be parallel (Anderson and Darling, 1964). 11
During pre-insemination coiling or probing of the male in
Rhabditis teres (Chuang, 1962), Pelodera teres (Jones, 1966),
Cylindrocorpus spp. (Chin and Taylor, 1969), Rhabditis pellio (Somers,
Shorey and Gaston, 1977), the spicules were periodically protruded to make contact with the cuticle of the female. Such a periodic extension of the spicules has not been observed in other species of nematode.
Mueller (1930) reported that during copulation in Ascaris lumbricoides the spicules were withdrawn into their sheaths and he thought that they played no part in insemination. However, this was based on fixed worms found in the coital position and more recent work on the copulatory behaviour of living specimens of several nematode species has shown that the spicules remained inside the vagina during the whole process of insemination, no matter how long this process took (Chuang, 1962;
Greet, 1964; Anderson and Darling, 1964; Jones, 1966, Sommerville and
Weinstein, 1964; Fisher, 1972; Chin and Taylor, 1969; Trudgill,
1976; Somers, Shorey and Gaston, 1977).
Formerly the spicules of most nematodes were considered to be involved in opening the female gonopore for copulation and were not considered as intromittent organs. The exception being Proleptus obtusus where one spicule was thought to be specially modified to act as an intromittent organ (Mueller, 1925). However, electron microscope studies on the spicules of Heterodera spp.(Clark, Shepherd and
Kempton, 1973), Nippostrongylus brasiliensis (Mclaren, 1976; Croll and Wright, 1976), Pratylenchus penetrans (Wen and Chen, 1976) and
Hoplolaimus galeatus (Hogger and Bird, 1974) have shown that the 12
shape of the spicules ensured an enclosed tubular structure and that the sperm passed through it into the female.
The duration of copulation has been found to vary considerably between species and also between individuals of the same species. Chuang (1962) noted that copulation took from a few minutes to a few hours in R.teres while in Panagrolaimus rigidus it was reported to vary from 15 seconds to 30 minutes (Greet, 1964). In
D. destructor copulation was observed to be completed within a few seconds (Anderson and Darling, 1964). In Cylindrocorpus spp. incomplete copulations were seen which lasted 10 - 15 seconds (Chin and Taylor,
1969), while in Xiphinema diversicaudatum (Trudgill, 1976) copulation
only lasted 4 -5 seconds before the female broke away with sperm continuing to flow from the male. Recently Somers, Shorey and Gaston
(1977) during their studies on R. pellio had shown that the mean time
that elapsed between first contact of the sexes and their ultimate
separation was 23.2 minutes, however, only 5.0 minutes were required for actual copulation.
In some parasitic species copulatory behaviour has been found to be greatly modified. In members of the family Syngamidae, nematodes which live in the tracheae and bronchi of mammals and birds,
the male and female remain permanently in copula (Smyth, 1976), while
in Trichosomoides spp. the male lives inside the female (Little and
Orihel, 1972). Males of an Anatrichosoma sp. insert part of the
posterior end of the body into the uterus of the female during
copulation and these have been suggested to be more primitive than
Trichosomoides spp. (Little and Orihel, 1972). 13
Trudgill (1976) has mentioned that when the inner surface of the tail of a "sexually aroused" male X. diversicaudatum touched another member of the same species, the male rapidly reversed and coiled its tail around the second nematode. These males seemed to be prepared to encircle another nematode at any point along its body and would attempt to copulate with females, larvae and other males. Similar observations were made by Chuang (1962) on R. teres, where males sometimes tried to copulate at the wrong place on the female leaving their spicules embedded in the cuticle of the female.
Male R. teres were also seen attempting to copulate with other males, where they inserted their spicules into the cuticle and released their sperm.
Anderson and Darling (1964) found that the anterior end of male D. destructor first touched the vulva of the female and then twisted away, probing further along the body. This process was repeated several times before insemination. Similarly, Jones
(1966), Somers, Shorey and Gaston (1977) and Chin and Taylor
(1969) observed in P. teres, R. pellio and Cylindrocorpus spp. respectively, that the anterior region of the male was usually the first part to make contact with female, although Jones was of the opinion that the bursa was the main sensory structure involved once the female was found. In P. rigidus (Greet, 1964) and X. diversicaudatum (Trudgill, 1976) coiling only took place when the posterior region of the male came into contact with the female.
Recent transmission electron microscope studies on the 14
spicules of Heterakis gallinarum (Lee, 1973), N. brasiliensis
(Lee, 1973; Croll and Wright, 1976; Mclaren, 1976), P. penetrans
(Wen and Chen, 1976), Heterodera (Globodera) rostochiensis (Clark,
Shepherd and Kempton, 1973), Syphacia obvelata (Dick and Wright,
1974), Aphelenchoides blastophthorus (Clark and Shepherd, 1977),
Dipetalonema viteae (Mclaren, 1976) and Necator americanus (Mclaren,
1976) have shown nerve processes running the length of spicules and these were thought to be involved in some sensory function. One or two pores have also been found at the tip of each spicule in
A. blastophthorus (Clark and Shepherd, 1977); P. penetrans (Wen and Chen, 1976) and Heterodera spp. (Clark, Shepherd and Kempton,
1973) and the presence of nerve endings beneath these pores suggested
a chemosensory function. It has been suggested that the sensillae near the tip of the nematode spicule aid in penetration of the vulva
without damaging the female (Clark, Shepherd and Kempton, 1973;
Anya, 1976).
Further evidence of a sensory function for the spicules has
-come from the demonstration of esterase activity in the spicules of
A. lumbricoides (Lee, 1962); Meloidogyne javanica and M. hapla
(Bird, 1966); H. gallinarum and N. brasiliensis (Lee, 1973);
some of this esterase activity may prove to be due to acetylcholin-
esterases, which would be indicative of cholinergic neurotransmission
(Wright and Aswan, 1976).
It seems reasonable to assume that the sensillae at the
ends of the spicules also aid in the detection of the vulval opening
and may even trigger ejaculation of sperm (Lee, 1973); it is also 15
posSible that they may be involved in sex attraction (Samoiloff,
McNicholl, Cheng and Balakanich, 1973).
Although the head region is often the first part of the male nematode's body to make contact with the female, a tactile response of the caudal region of the male was found to be necessary for coiling to occur, suggesting the possible importance of the caudal papillae in copulation (Chitwood and Chitwood, 1950;
Lee and Atkinson, 1976).
The structure of the caudal papillae has not been
examined closely in many species of nematode. Goldschmidth (1903) reported that the genital papillae of A. lumbricoides consisted of
raised areas of the cuticle perforated by a canal containing one to
three nerve fibres surrounded_by supporting cells. Chitwood and
Chitwood (1950) noted that the nerve fibres innervating the pre-
cloacal papillae of A. lumbricoides eventually reaches the ventral
nerve, while those innervating the post-cloacal papillae reached
the latero-caudal nerves. In Aspiculuris tetraptera Anya (1973a)
reported the possible presence of the putative neurotransmitter
5 - hydroxytryptamine in cells associated with the genital papillae
on either side of the cloacal opening and also in the rectal ganglion
to which the papillae are connected.
The caudal papillae have recently been examined with the
electron microscope in S. obvelata (Dick and Wright, 1974),
D. viteae (Mclaren, 1972) and A. blastophthorus (Clark and
Shepherd, 1977). A central nerve process opening to the exterior
was found in D. viteae and A. blastophthorus but not in S. obvelata; 16
the presence of a pore in the former two species suggesting a chemosensory function for the caudal papillae in addition to a possible mechanosensory one.
Finally, it has been reported that mutant strains of male
Caenorhabditis elegans which are deficient for the putative transmitter dopamine in six neurons in the tail show some loss of mating efficiency, requiring a higher density of older, more receptive hermaphrodites
(Sulston, Dew and Brenner, 1975).
Sex attraction in nematodes
Since Greet (1964) demonstrated the phenomenon of sex
attraction in the free-living nematode P. rigidus sex attraction has
been found in 20 other species of nematode from different habitats.
In each of the nematode species studied the female nematode has
been shown to attract the male but the reverse phenomenon has only
been found in P. rigidus (Greet, 1964), Trichinella spiralis
(Bonner,and Etgas, 1967), Camallanus sp. (Salm and Fried, 1973) b and A. tetraptera (Anya, 1976). It has also been found that the
attractant was somewhat species specific and water soluble. There
are no reports of homosexual attraction in nematodes.
The main differences of opinion concerning sex attraction
have centred around the time the female starts and stops secreting
attractant and the control mechanism involved, whether both sexes
attract each other, and which organs produce the sex attractant. 17
Some workers have tried to discover the origin of the sex attractant in nematodes. Green and Greet (1972) working with
Heterodera spp. suggested that the attractant was produced in the hypodermis and was secreted all over the body; although in the case of H. schachtii more attractant appeared to be secreted from
the tail region. However, these authors also quoted Doncaster as suggesting that secretions from the vulva were necessary for sex attraction to occur. There is also evidence for the secretion of a sex attractant from the vulva in N. dubius, where it was found that males failed to show bursal flaring when the female's vulva was blocked with silicone grease (Merchant, 1970). Recently, b Anya (1976) has reported that male A. tetraptera were more attracted
to material originating from the female's reproductive organs than from other parts of the female body. He proposed that the sex attractant in female A. tetraptera was produced by certain secretory cells located in pulvillus (a cushion-like group of cells in the
post-anal region), while female A. tetraptera were more attracted
towards material from the caudal gland region of the male.
Samoiloff (1970) on the other hand, suggested that the attractive substance in Panagrellus silusiae was derived from dissolved cuticular material produced during the final moult of the female.
Cheng and Samoiloff (1971) produced evidence that fourth stage larval females of P. silusiae could attract adult males and that fourth stage larval males responded to adult females. Similarly,
Windrich (1973) maintained that fourth stage larval females of
Ditylenchus dipsaci could attract males. However, Cheng and 18
Samoiloff (1972) while studying the effect of cyclohexamide and
hydroxyurea on mating behaviour and gonadogenesis in P. silusiae
found that larval females treated with hydroxyurea, an inhibitor
of DNA synthesis, did not develop a mature reproductive system and
the adult males and females were not attractive to each other.
It was thus proposed that sexual attraction depended upon the
complete development of the reproductive system. Samoiloff,
McNicholl, Cheng and Balakanich (1973), on the basis of laser
microbeam irradiation of the tail region of P. silusiae, have
claimed that the receptor for mating attraction was situated in
the spicules.
Female copulatory activity in nematodes
During a study on the reproductive behaviour of
N. dubius in vivo Sommerville and Weinstein (1964) observed that
after moulting the female stored some oocytes in the distal region of
the ovary at the junction with the uterus. The number of oocytes accumulating in this region increased in the following few days and some passed into the uterus. Similar observations were made by
Phillipson (1969) in N. brasiliensis and he concluded that the presence of unfertilized eggs in the uterus of the female was not essential for the copulation to occur. Johnson, Orihel and Beaver
(1974) also found that unfertilized oocytes were stored in the growth zone of the ovary in D. viteae and that eventually they passed through 19 •
the oviducts into the uterine branches; they also observed that eggs (oocytes) reaching the uterus prior to insemination were not subsequently fertilized and gradually degenerated.
Fisher (1972) has studied the effect of delayed mating on the fecundity and life span of female Aphelenchus avenae. He observed that although the life span of the female was increased from 18 days to 34 days, delayed mating reduced the total number of eggs laid from 134 to 83. Similarly, Gaston, Shorey and.
Platzer (1974) observed that the life span of virgin females of a
Rhabditis sp. was 12 1: 0.7 days compared with 10.8 0.6 days in once-copulated females and 7.4 - 0.4 days in regularly copulating females.
There is little information available on the timing of the first copulation in nematodes. The first copulation of female V. brasiliensis did not apparently occur until at least
104 hours after rats had been subcutaneously infected (i.e. after about 8 hours from the final moult); the majority of nematodes copulating for the first time approximately 120 hours after
infection (Phillipson, 1969). While Johnson, Orihel and Beaver
(1974) reported that mating usually occurred soon after the final
moult of female D. viteae. In H. rostochiensis females„the minimum
time taken for the first copulation to occur was found to be 24 days
after the roots were invaded by the second stage larvae; thereafter
the number of females which copulated steadily increased and nearly
all the females were mated 45 - 50 days after inoculation (Evans,
1970). 20
Phillipson (1969) examined the rate of sperm depletion in normally copulated female N. brasiliensis of varying ages. He observed that 5 - 20 day old females stopped passing fertile eggs after 5 - 12 days of their re-inoculation. He also observed that in old females sperm consumption was slow compared to young females and he concluded that normal females must copulate once every two days in order to maintain fertile eggs output. Somers,
Shorey. and Gaston (1977) also concluded similarly for R. pellio.
Similarly, sperm were depleted after 14 - 85 days in once-copulated female D. viteae (Johnson, Orihel and Beaver, 1974), and it was suggested that frequent mating was required for continuous egg production. It was thought that the genital girdle in
A. lumbricoides was formed as a result of the diminishing store of sperm or the increasing number of unfertilized eggs in the uterus; after copulation the girdle involution was found to be relatively rapid (Beaver and Little, 1964).
Female D. destructor have been shown to remain receptive to males for about a week after the final moult and a single female could copulate several times during this period with more than one male, although several hours usually elapsed between two successive copulations (Anderson and Darling, 1964). Greet (1964), on the other hand, often observed two successive copulations by female P. rigidus with different males.
Phillipson (1970) found that unisexual infections of adult female N. brasiliensis between one day and 3 months old always copulated rapidly when mixed with males. Isolated females of 21
H. rostochiensis remained fertilizable for at least 40 days after
the time of their normal first copulation, although fewer females
were fertilized after 70 days than after 35 days (Evans, 1970).
Phillipson (1969) reported that normal female
N. brasiliensis produced about 1000 fertile eggs/day and that the
rate of egg production remained steady until the onset of the host's
immune response. On average female N. brasiliensis produced 3500
fertile eggs without further insemination. Female A. avenae were
.found to lay 478 -.579 eggs in 9 - 18 days, egg production following
a skewed normal distribution (Fisher, 1972). • Egg production has also been examined in several other
nematode species. Female Ditylenchus myceliophagus were found to
receive about 40 sperm during the first copulation, which occurred
just after the final moult, and after laying about 40 eggs the
female could be re-inseminated laying a further 20 eggs (Cayrol,
1970). D. dipsaci females layed 207 - 498 eggs in two batches in
45 - 73 days on onion seedling (Yuksel, 1960), while P. rigidus
laid up to 344 eggs in 35 days at 20°C (Mianowska, 1970), and
Pelodera strongyloides an average of 472 viable eggs in ten days
at 17°C (Nigon, 1949). Female Rhabditis maupasi were observed to
live for 7 - 10 days after reaching maturity and to lay 150 - 300
eggs, the life of the adult male was only about one third that of
the.female (Otter, 1933). In a more detailed study, on
Pristionchus aerivora, Merrilland Ford (1916) found that adult
females usually lived for 12 - 13 days after commencing to lay
eggs. During a period of 13 days one female was found to copulate 22
with seven males, and deposited 317 fertile eggs and 14 infertile eggs. Males of P. aerivora were somewhat less numerous than females, they lived for about 19 days and copulated on average with 10 females.
Females of Pheidole pallidula and Hexamermis sp. reared in the absence of males failed to lay eggs (Vandel, 1934) and some lived for 22 - 33 months after moulting, whereas females allowed to copulate and which layed eggs lived for only about 5 months.
Histamine has been suggested to play a role in egg production in Ascaris suum, where it was found in the reproductive system, as immature adults contained less histamine than mature egg-laying females (Phillips, Sturman and West, 1975). Histamine has also been found in C. elegans (Pertel and Wilson, 1976).
However, in A. avenae exogenous histamine principally affected stylet thrusting and not vulval contractions and increased oviposition as was the case with some other biogenic amines, adrenaline and 5 - hydroxytryptamine (Croll, 1975).
Male copulatory activity in nematodes
The sexual capacity of male N. brasiliensis was examined by Phillipson (1970) and his results indicated that a male could inseminate on average one female every two hours during the first
15 - 20 hours of their adult life. Males of D. destructor were found to remain sexually active for at least three weeks after the 23
final moult (Anderson and Darling, 1964), while D. dipsaci
(Yuksel, 1960) and H. (G.) rostochiensis (Evans, 1970) remained sexually active for 45 - 73 and 9 - 10 days, respectively.
Fisher (1972) observed that males of A. avenae copulated 18 to
26 times in 5 weeks at 27°C, and he noted that the interval between two successive copulations appeared to increase with age of the male.
Recently Somers, Shorey and Gaston (1977) also studied the reproductive potential of male R. pellio and found that they remained sexually active up to 9 - 10 days although their mean number of matings per day decreased with increasing age.
The'vas deferens is a major region of the male reproductive tract in nematodes which obviously plays an important role in reproduction, although very little is known about its physiology or biochemistry. It has been shown to be differentiated into two or three regions in nematodes, each with characteristic secretory cells
(Chitwood and Chitwood, 1950; Anya, 1966; Hulinska, 1973; Shepherd and Clark, 1976) which produce secretions differing in their chemical composition and probably in their function (Anya, 1966; Hulinska,
1973). Serotonin (5 - hydroxytryptamine) and possibly other indolealkylamines are thought to be present in the male reproductive tract of A. tetraptera (Anya, 1973a, b), and it has been postulated that such biogenic amines initiate contraction of the female's uterus, forcing the sperm into the seminal receptacle. A similar function had previously been suggested for 5 - hydroxytryptamine in the reproductive system of some other invertebrates (Mann, 1960, 1963). Exogenous 24
serotonin, 5 - hydroxytryptophan, and epinephrine (a6naline) have
been shown to induce rapid and prolonged contraction of the vulva in
C. elegans, A. avenae and P. redivivus. Serotonin also caused ventral
bending of the male tail with extrusion of the spicules in P. redivivus
without ejaculation occuring (Croll, 1975). While Foor and McMahon
(1973) found that homogenates of the vas deferens of A. suum initiated
pseudopodial formation in sperm in the seminal vesicle.
Nematode sperm have been observed to become motile and undergo
marked morphological changes after they enter the female reproductive
tract (Poor, 1970). In particular smooth endoplasmic reticulum
rapidly appears in the cytoplasm of the sperm and there is a fusion of
certain membranous elements in the cytoplasm with the plasma-membrane.
With the exception of A. tetraptera (Lee and Anya, 1967) all nematode
sperm examined so far at ultrastructural level have these characteristic
membranous organelles.
Various hydrolytic enzymes notably adenosine triphosphatase,
esterases and acid phosphotases, have been detected in the sperm of nematodes (Lee and Anya, 1967; Clark, Moretti and Thomson, 1967;
Lee, 1971). In A.•lumbricoides (Clark, Moretti and Thomson, 1967) and H. gallinarum (Lee, 1971) acid phosphatase activity was associated with the membranous organelles. Lee and Leston (1971) also found succinate dehydrogenase activity in the sperm of H. gallinarum, while Anya (1966) identified alkaline phosphatase, adenosine triphosphatase, non-specific esterases, acid phosphatase, glucose -
6 - phOsphatase and cholinesterase activity in the semen of
A. tetraptera. 25
Foor (1970) observed that after insemination, when most of the membranous organelles had fused with the plasma-membrane and released their contents, the size of the sperm seldom exceed more than
50% of their initial volume. Similarly, Phillipson (1969) observed that sperm in N. brasiliensis measured 17.8 pm in diameter in the seminal vesicle of the male but only 15.1 pm in diameter in the seminal receptacle of the female. The fusion of the membranous organelles after insemination and the release of secretory material may be involved in sperm movement -and in the ageing of the sperm in the uterus of the female, together with their loss of fertilization ability. This secretory material may also affect the further copulation of the female.
Sex ratio in nematodes
The sex ratio is known to be largely influenced by the environment in parthenogenetic and hermaphroditic nematodes
(Cobb, Steiner and Christie, 1927; Caullery and Comas, 1928;
Christie, 1929; Nigon, 1949; Tyler, 1933; Triantaphyllou,
1960, 1971; McClure and Viglierchio, 1966; Davide and Triantaphyllou,
1968). The detailed studies made by Nigon (1949) as an adjunct to his studies of sex determination, showed that in some bisexual, amphimictic nematode species the sex ratios were nearly equal. Males were found to be about 48% of adult nematodes in Pelodera strongyloides (Rhabditis strongyloides), 43% in P. lambdiensis (R. lambdiensis), 34% in
Pristionchus robustus (Diplogaster robustus) and 52% in P. rigidus. 26
In P. strongyloides the mean % of males did not differ significantly
at 13°C, 17°C or 24°C, nor was there any significant difference
between the progeny produced by a female on different days of its
reproductive life (46.6 - 49.5% males over 20 days at 13°C).
Similar results were obtained with Pristionchus ltherithieri.
The wide fluctuations in the male : female ratio in various
amphimictic bisexual Heterodera species have been attributed to the
inability of the female larvae to develop to maturity under conditions
of stress (Kerstan, 1969). It was shown that equal numbers of male
and female H. schachtii developed in plants infected by single larva
and that the increase in the male : female ratio under conditions of
crowding or other adverse conditions was caused by the reduction in
the numbers of developing females rather than an increase in the
number of males. A similar conclusion was reached by Johnson and
Viglierchio (1969) and it appeared that the sex ratio in these
Heterodera species was genetically controlled and sex expression was
not modified by environmental influence only the sex ratio is.
Hechler and Taylor (1966) found a male :female ratio of
between 1 : 7 and 1 : 2.5in Seinura celeris populations. Ellenby and
Smith (1966) found an average male : female ratio of 1 : 3.4 in
P. redivivus in wholemeal wheat flour, whereas Hansen and Cryan (1966) found that it was 1 : 1 in axenic cultures. 27
C ONCLUSIO'N S
From the above review of copulatory behaviour and reproductive biology of nematodes it is apparent that information is lacking in many areas.
Previous work on the functional maturation of nematode sperm have been based largely upon transmission electron microscope studies and such studies have provided little information on the behaviour of sperm in vivo and were also subject to artifacts due to fixation and embedding. The aim of the present work was to examine the behaviour and morphology of living sperm of P. redivivus before and after insemination.
It was also clear that while the spicules and probably the caudal papillae aid in copulation in nematodes, little of the work described combined structural observations with behaviour studies.
In the present work the posterior region of male P. redivivus was examined under both the light microscope and the scanning electron microscope, and the general copulatory behaviour of the male observed in order to have a better understanding of the role played by such organs in copulation.
In regard to the copulatory behaviour of male nematodes, while this has been described in general terms in many nematodes little attempt has been made to quantify such behaviour during the life span. Similarly, a comparative study of virgin and normally copulating females has not been made in any detail.
Sex attraction in nematodes has shown that males are attracted 28
to the adult females and that sex attractants are probably produced
in the female reproductive system but the effect of copulation on
sex attraction has not been studied. In the present work an attempt
was made to see if copulation had any effect on sex attraction in
P. redivivus.
Finally, although there is plenty of information about the
life span and fecundity of female nematodes relatively little is
known about their mating requirements. In this work an attempt was
made to determine some of the major factors involved in regulating
copulation in female P. redivivus.
For the sake of convenience the results are presented in
five experimental sections. The first section deals with the male
and female reproductive organs, the process of copulation and the functional maturation of the sperm in P. redivivus. In the second
section the copulatory behaviour of male P. redivivus during their
life span is described. The next two sections deal with the effect of copulation on the development of the female's reproductive organs and on sex attraction respectively, and in the last section some of the factors influencing the female's copulatory behaviour are described.
In the present work the following terms are defined as follows:
Copulation. This is regarded as the active act of transferring semen
(including sperm) from the male reproductive system to the female 29
reproductive system. This starts with the penetration of the spicules into the vagina of the female and finishes as soon as spicules are withdrawn. Copulation was considered incomplete when the spicules penetrated the vagina but insemination did not occur.
Libido. This was a measure of the ability of male nematodes to copulate within a fixed period of time, and was expressed asia % of males copulating.
Attraction. The ability of nematodes to induce other nematodes to move towards them.
Attractiveness. The degree of attraction.
Response. The ability of nematodes to be attracted by and move towards other nematodes.
Responsiveness. The degree of response. 30
SECTION I
The experimental animal and its culture in the laboratory
I.A. Panagrellus redivivus (Goodey, T., 1945), Order Rhabditida
(Oerley, 1880), Family Panagrolaimidae (Thorne, 1937).
P. redivivus is a microphagous, free-living soil nematode found also in fermenting media. It is dioecious and viviparous and its reproductive and post-embryonic development have been described by
Hechler (1970). The distribution of the genus and details of its taxonomy are given by Hechler (1971) and a classification of the genus by Goodey
(1963). The experimental culture was obtained from the one maintained by
Dr. D.J. Wright at Imperial College since 1971; previously this culture was maintained at Newcastle University, Zoology Department (from 1963).
I.B. Monoxenic culture
The culture was brought into the monoxenic state (Dougherty,
1959) using the procedure described by Cryan, Hansen, Martin, Sayre and
Yarwood (1963). The treated nematodes were placed on Nigon's agar
(Nigon, 1949) in sterile disposable plastic petri dishes which had been inoculated with Escherichia coli 12 hours prior to the addition of the worms. This monoxenic culture was maintained at 25°C. 31
I.C. Preparation of agar plates
Czapex dox agar (oxoid), Nutrient agar (oxoid) and Nigon's agar (Nigon, 1949) were all used to culture P. redivivus. Nigon's agar
(Table I) was selected in preference to Czapex dox agar or nutrient agar because it was difficult to maintain fungal-free plates on Czapex dox agar while bacterial growth was too rapid on nutrient agar resulting in a much slower rate of nematode reproduction. Therefore, Nigon's agar was used to culture the nematodes routinely. Nutrient agar was used to culture E. coll. All experiments were conducted on Standard
Davis Agar.
Table I
Nigon's Agar
MgSO4 7H20 0.75 gm.
K HP0 0.75 gm. 2 4 NaC1 2.75 gm.
KNO 3.00 gm. 3 peptone (bacteriological) 2.50 gm.
lecithin 1.00 gm.
agar 15.00 gm.
distilled water 1 litre 32
I.D. Observation on P. redivivus at low magnification (X 10, X 40)
Davis agar (1.5% W/V) was prepared in 50mm diameter sterile disposable plastic petri dishes. E. coli were placed on the agar in
the centre of the plate and spread over a 13mm diameter area with a fine bristle. Nematodes were then placed on the E. coli and observed
under the compound microscope.
I.E. Observation on P. redivivus at high magnification (X 100)
For these observations perspex slides were used 3.5 inches
X 1.5 inches and 0.2 inches thick, with a 0.5 inch diameter hole in the
centre of each slide (Fig.la). Nematodes were placed in a small drop of
tap water on a 22mm square glass cover-slip and each drop was covered with
a piece of solidified Davis agar (1.5 % W/V) prepared as follows:
Drops of moulten agar were placed on a clean micro-scopic
slide and allowed to solidify (Fig. lb) and the flattened drops were
then removed with a razor blade (Fig. lc) and each placed on top of
a drop of water containing a nematode (Fig. 1d).
Small pieces of Sellotape, 0.2 - 0.3 inches by 1.0 - 1.2 inches,
were then placed at the four edges of the cover-slip (Fig. le) and the
perspex slide pressed down carefully into the cover-slip so that the
agar drop was in the centre of the hole (Fig. lf). Another piece of
Sellotape 1.0 X 1.5 inches was placed over the other side of the hole
in the perspex slide forming an air-tight chamber. The slide was then
observed from the glass cover-slip side under oil immersion. 33
a
\0 0 0 0
F1\
d
e
f
Fig. 1 Apparatus used for high magnification observations on P. redivivus (see text). 34
I.F. Scanning Electron Microscopy
I.F.1. Tail region of adult male P. redivivus
Nematodes were washed several times in distilled water and then fixed in 2% (V/V) formalin for 24 hours at room temperature.
This method of fixation consistently resulted in the protrusion of the spicules. The specimens were then washed several times in phosphate buffer (0.1M, pH, 7.4) and dehydrated with acetone as follows: nematodes 3 in 0.1 cm of phosphate buffer were placed in a 30 X 10 mm cavity block and kept in a desiccator over dry acetone for 24 hours at room temperature; the buffer in the block being slowly replaced with acetone by vapour exchange. Dehydrated specimens were subjected to critical point drying in carbon dioxide (Hayat and Zirkin, 1973) and then mounted in the centre
of an aluminium stub with double-sided Sellotape. The Sellotape and stub were then carefully covered with colloidal silver with a fine brush.
Specimens were finally coated with gold using a Polaron Sputter Coater,
and examined in a Cambridge IIA Stereoscan Microscope. at 5 - 20 kv.
I.F.2. Sperm
Newly inseminated female nematodes were placed in a drop
of saline solution (Section II.E.2.) and each nematode was cut in
the middle with a disposable syringe needle. The floating chains of
sperm were picked up with a dropper and placed in the centre of a
13 mm diameter glass cover-slip. Another cover-slip was immediately 35
placed over the drop and the two cover-slips were held together by a drop of beeswax. The cover-slip "sandwich" was then pushed into a groove made out of a piece of sponge (Fig. 2). The whole preparation was then immersed in 2.4% (V/V) glutaraldehyde in
0.1M phosphate buffer (pH. 7.4 at 4°C) for 24 hours. The preparation was then washed in 0.1M phosphate buffer for 4 hours at
4°C and this was repeated three times with fresh buffer solutions.
The preparation was next dehydrated in the following sequence of acetone-water solutions: 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95% and 100% acetone, with 15 minutes in each concentration.
Following a transfer to fresh 100% acetone the cover-slips were gently removed from the groove and the two cover-slips separated; the sperm were then subjected to critical point drying and the whole cover-slip was fixed on an aluminium stub with double-sided Sellotape and coated with colloidal silver. The preparation was finally coated with gold and examined as in Section I.F.1.
Fig. 2 Handling device for Scanning Electron Microscopy of nematode sperm (x 0.75). 36
SECTION II
Observations on the general
reproductive biology of P. redivivus
II.A. The male reproductive system
P. redivivus has a monorchic type of reproductive system
(Hyman, 1951) the male having one testis, one seminal vesicle and one
vas deferens (Hechler, 1971, and Fig. 3). The adult testis has a short
reflexed portion at the anterior end but there are•no constrictions
to easily distinguish one region of the reproductive system from the
next. However, the distinctive cellular morphology of the different
regions can be used to identify specific parts of the reproductive
system in living worms. The reflexed region of the testis was found
to be the germinal zone in the closely related species Panagrellus
silusiae (Pasternak and Samoiloff, 1972); this region being followed
by a wider growth zone. Posterior to the growth zone is the seminal vesicle. This region was found to become very distinctive in
P. redivivus when large numbers of sperm were present giving it a
granular appearance; a band of 7 — 8 clear cells could also be seen around the seminal vesicle (Fig. 3). These clear cells have not been described previously and their function is unknown. All mature sperm were stored in the seminal vesicle before copulation. 37
Fig. 3 Diagrams showing general anatomy of mature adult male
and female P. redivivus (x 100). A, ventral view of
male; B, lateral view of female. Bubble-like structures,
BUB STRU; caudal papilla, CA PA; embryo, EMB; germinal
zone, GER Z; growth zone, GRO Z; globules, GLO;
gubernaculum, GUB; larva, LAR; oocyte, 00CY; ovary,
OV; post-vulvar uterine sac, PVUS; band around seminal
vesicle, SEM BAND; seminal vesicle, SEM VES; sperm, SP;
spicule, SPI; vas deferens, VAS DEF. 38
A B 39
Between the seminal vesicle and the vas deferens aggregates
.of small globules were often found. These structures have not been
described previously and their origin and function is as yet unknown
(Fig. 3); they appeared to increase in number as the interval between
two successive copulations increased.
The vas deferens is the largest and most posterior part of
the reproductive tract (Hechler, 1971) and this region appears to have a a secretory function in nematodes (Anya, 1976); it can be distinguished
from the seminal vesicle in P. redivivus by its clear, non-granular
appearance. Most of the vas deferens in P. redivivus was found to be
composed of relatively large cells with smaller cells at the anterior
end. Near the posterior end of the vas deferens a small group of
bubble-like structures were seen which had not previously been described;
these passed into the uterus of the female during copulation (Fig. 3),
similar structures redeveloped in the vas deferens within 1 - 2 hours
of copulation. The importance of these bubble-like structures during
or after copulation was not known, but like the globules found between
the seminal vesicle and the vas deferens changes occurred with increasing
intervals between copulations, the bubble-like structure increasing in
size with time.
Posterior to the glandular vas deferens there is a short
non-glandular region of relatively small cells known as the
ejaculatory duct (Pasternak and Samoiloff, 1972). However, the
involvement of this duct in ejaculation of semen during copulation
has never been observed. 40
II.B. Male accessory reproductive organs
II.B.1. The Spicules
Male P. redivivus have two spicules of equal size and shape which are ventrally curved (Hechler, 1971, and Figs. 4, 6 and 7). The manubrium of each spicule is hooked and there is one opening on its dorso-lateral side for muscle attachment and nervous innervation. A thin, membranous velum arises just behind the proximal end of - the spicule and extends to the distal end (Hechler, 1971 and Figs. 4 and 5).
Each velum was found to form part of an incomplete tube when both the spicules were extended (Figs. 6 and 7). The distal end of each spicule is seclerotized on the dorsal and ventral sides, but most of the tip is unhardened. The present light microscope studies have shown that the distal end of each spicule has a small pore on both the dorsal and ventral seclerotized sides (Figs. 4 and 5).
II.B.2. The Gubernaculum
This is a small shallow trough of varying thickness which proximally encloses the spicules on the dorsal side only, and distally encloses the spicules on the dorsal and lateral sides (Hechler, 1971).
The gubernaculum appears to be composed of similar material to the spicules; it has muscular attachments at its proximal end (Hechler, 1971;
Chitwood and Chitwood, 1950). 41
Fig. 4 Light micrograph of spicules of male P. redivivus showing membranous vela and distal tips (x 1800). 42
B
Fig. 5 Diagrams of spicules and gubernaculum of male P. redivivus (x 2000). A, lateral view; B, ventral view. Gubernaculum, GUB; manubrium, MANU; pore, P; spicule's tip, SPI TIP; velum, VEL. 43
II.B.3. Caudal Papillae
There are seven pairs of caudal papillae on the posterior end of male P. redivivus (Hechler, 1971). One pair of papillae are pre-anal, two pairs are ad-anal and four pairs are post-anal. Two pairs of post- anal papillae are on the sub-dorsal side; the other caudal papillae are sub-ventral. There is also a single ventro-median caudal papilla just anterior to the cloaca (Hechler, 1971). These papillae are shown in the scanning electron micrographs in Figs. 6 and 7.
II.C. The female reproductive system
The female of P. redivivus has a single set of reproductive organs like the male (Hechler, 1971). The vulva is a transverse slit situated mid-ventrally, anterior to the anus (Fig. 3), and the vagina and uterus run anteriorly. The vagina is a short, highly muscular region flattened dorso-ventrally. The lumen of the vagina is straight except for a protrusion of the anterior wall and a corresponding notch in the posterior wall just within the vulval region. During copulation the male spicules were sometimes observed to lodge in this notch and thus fail to penetrate deeply into the vagina. The cuticle lining the lumen of the vagina is relatively thick.
A dorsal wall with lobe-like thickening is present between the openings to the uterus and the post-vulvar uterine sac; the latter is generally accepted to be a vestigeal uterus (Chitwood and Chitwood,
1950) and it joins the vagina on the dorsal side by a transverse slit. 44
Fig. 6 Scanning electron micrograph of spicules of male P. redivivus (ventro-lateral view) showing channel formation by the membranous vela and release of sperm (x 3600).
Fig. 7 Scanning electron micrograph of posterior end of male P. redivivus (dorso-lateral view) showing caudal papillae and spicules (x 2000). 45
The uterus is a long, extendable tube running up to the posterior bulb of the oesophagus. There are faint striae marking the whole length of the uterus. Two sphincters at the flextum separate the seminal receptacle from the uterus and the oviduct (Hechler, 1971 and Fig. 3).
In fact, sperm were not found to be stored in the seminal receptacle of P. redivivus but rather it was found to act as a double valve between the oviduct and the uterus, through which oocytes slowly passed into the uterus. Fertilization probably takes place in the seminal receptacle.
The ovary is relatively long and generally extends posteriorly to the post-vulvar uterine sac, and sometimes it may extend into post-anal region. The ovary is connected to the seminal receptacle via a small oviduct which usually contains a few maturing oocytes (Hechler, 1971, and Fig. 3). However, if copulation was delayed mature oocytes were found to accumulate in the oviduct which resulted in its considerable elongation and loops formed to accomodate its length inside the pseudocoel sometimes pushing the seminal receptacle ventrally towards the uterus.
II.D. General copulatory behaviour of P. redivivus
II.D.1. Pre-insemination coiling of the male
When the posterior end of the male touched the female's body, the male sometimes coiled around the female. This appears to depend primarily on the male's physiological condition as coiling only 46
occurred when sperm were present in the seminal vesicle and when there were bubble-like structures in the posterior part of the vas deferens (Section iI.A.). Males which had just copulated did not coil around another female, irrespective of the female's reproductive state. When the cloacal region of a physiologically mature male touched the female's body coiling took place (Fig. 8), probably with the aid of sensory organs in the spicules and caudal papillae
(see for example, Mclaren, 1976). The initial place of coiling was not important, males being found to coil around the oesophageal, middle, or post-vulvar regions of the female. During coiling the male was observed to move backwards around the female and in most cases the male kept its spicules in the dorsal or ventral line of
the female. Normally a male had only one coil of its body around the female, but if it had been separated from a female for a number of hours it often formed two or three coils (Fig. 9). During coiling the
male exerted considerable pressure on the female's body.
As the male coiled around the female the female moved slowly
backwards and forwards through the coil(s) and if the spicules of the
male touched the vulva penetration of the spicules into the vulval
opening usually occurred. If the spicules did not come into contact
with the vulva the male eventually uncoiled its posterior end.
Uncoiling also took place when the male's coils reached the tail
region of the female, probably due to the thinness of the tail.
Males were never found following escaped females.
Males physiologically ready to copulate were found to coil
as readily around gravid females as around virgin females and also 47
Fig. 8 Typical position of male and female P. redivivus immediately prior to pre-insemination coiling. Bubble-like structures, BUB STRU; post-vulvar uterine sac, PVUS; spicule, SPI; uterus, UP. 48
Fig. 9 Coiling of an isolated male P. redivivus around female. Larva, LAR; spicule, SPI. 49
coiled around other males, larvae or even sometimes their own bodies.
Occasionally several males were found coiling around the same female, especially when the female was a newly moulted virgin. During the process of pre-insemination coiling both male and female continued
to feed.
II.D.2. Penetration
When the spicules touched the vulva the male thrust them into the vagina. Sometimes the female moved out of the coil before the spicules penetrated deeply into the vagina, but in virgin females or in females which had very few sperm in the seminal receptacle the spicules generally penetrated normally; about 2/3 of the total length of the spicules going into the vagina. Full penetration of the spicules took place within a few seconds and their tips extended into
the uterus. Male P. redivivus were never found trying to penetrate other regions of the female's body with their spicules, unlike some other nematode species (Chuang, 1962). The male stopped feeding and moving as soon as the spicules started to penetrate into the vagina, whereas feeding and movement in the female were not affected.
II.D.3. Insemination
Insemination took place once only per copulation immediately after the full insertion of the spicules (Fig. 10). The origin of the propulsive force for insemination was not located but presumably started 50
Fig. 10 Position of male and female P. redivivus during insemination. Sperm, SP; spicule, SPI. 51
anterior to or in the region of the seminal vesicle, forcing seminal fluid into the vas deferens. Local pressure may also have been exerted by the shortening of the male's body as the anterior region of the male was observed to shorten during insemination.
During insemination a clear fluid, most probably a secretion of the vas deferens, passed into the female together with the bubble- like structures previously described (Section II.A.), entering the uterus and post-vulvar uterine sac. This material was followed by the seminal fluid which had a granular appearance, quite distinct from the fluid of the vas deferens. The bubble-like structure from the posterior region of the vas deferens passed into the post-vulvar uterine sac, or anteriorly into the uterus where they rapidly disappeared.
Almost all the sperm which entered the vas deferens passed into the uterus of the female. Semen (seminal fluid and secretions from the vas deferens) sometimes reached the mid-region of the uterus due presumably to the high pressure developed in the male reproductive tract during insemination. In the newly inseminated females the sperm appeared as a semi-solid mass but they became more distinct after a few minutes, probably due to the absorption of the semen by the uterus.
About 20 - 30 sperm were normally transferred during insemination.
II.D.4.- Insemination in the post-vulvar uterine sac
During insemination the post-vulvar uterine sac generally received fluid from the vas deferens which was eventually absorbed by the surrounding 52
cells or which passed into the uterus. When the spicules do not penetrate fully into the vagina seminal fluid sometimes went into the post-vulvar uterine sac. Seminal fluid sometimes also passed into the post-vulvar uterine sac during the vaginal contractions which usually occurred soon after insemination, particularly when a large number of sperm were received. During vaginal contraction exchange of fluid between the uterus and the post-vulvar uterine sac took place probably due to differences in hydrostatic pressure.
The male started feeding after insemination even if it was still coiled and the spicules were still inside the vagina. Both virgin and non-virgin females continued to feed during and after insemination, except for newly moulted females receiving more than
30 sperm (from a long-isolated male); these females sometimes stopped feeding for some time.
II.D.5. Post-insemination coiling of the male and its attachment
to the female
In most cases the process of penetration and insemination was completed within one minute. The degree of post-insemination coiling depended upon both the male and female. If the female started to move immediately after insemination then the male was forced to withdraw its spicules and uncoil its posterior end from around_the female. However, if the female did not move then the male sometimes remained in a coiled position for up to 2 - 3 minutes (Fig. 10).
Sometimes a female was found to rotate the posterior half of its body 53
longitudinally, loosening the grip of the male coils and allowing
the female to move away. In most of these cases the male was unable
to withdraw its spicules before the female forcibly moved out of the
coil leaving the male's spicules extruded, these were later withdrawn
by the male.
In some cases males were found to be still attached to the
female's body even after they had withdrawn their spicules and uncoiled
their posterior ends. This was usually where post-insemination coiling
had been ptolonged and this may have been due to suction pressure
(Fig. 11); this type of attachment usually lasted for 1 - 2 minutes,
but lasted up to 15 minutes in a few cases. Both male and female
nematodes could be seen trying to detach themselves and this type of
attachment appeared to be only broken by considerable force. The
male moved away immediately after detachment from the female.
II.E. Maturation of sperm in the female
II.E.1. Introduction
It is now well established that the sperm of most nematode
species only become functionally mature inside the uterus of the
female (Foor, 1970). However, the nature of the sequence of the
changes that occur in the sperm are not well described. Present
knowledge of the morphological changes taking place in nematode
sperm are based upon ultrastructure studies and these have the
disadvantage that artefacts may appear during the processes of fixation 54
Fig. 11 Position of male and female P. redivivus during post- insemination attachment. Sperm, SP; spicule, SPI; post-vulvar uterine sac, PVUS. 55
and embedding. Earlier findings suggested that nematode sperm were normally motile inside the female uterus and attempts were made by several workers to observe living sperm in vitro although these met with little or no success (Foor and McMahon, 1973; Lee and Anya, 1967;
Shepherd, Clark and Kempton, 1973; Somerville and Weinstein, 1964).
The purpose of the present study was to observe living sperm of P. redivivus in a balanced saline solution after different time intervals and from different parts of the uterus, and to note changes in the sperm morphology.
II.E.2. Experimental Procedure
Nematodes of different reproductive states were carefully selected from the standard laboratory culture on Nigon's agar and placed in a drop of saline solution the composition of which is shown below:
gm/litre molarity
NaC1 4.51 gm 0.0772• .
KC1 0.12 gm 0.0016
0.12 gm 0.0011 CaC12 MgC12.6H20 0.02 gm 0.0001
Na SO 0.03 gm 0.0002 2 4 This solution was simply a 1 : 3 dilution of earthworm Ringer
(Pantin, 1964), which was found by trial and error to be the most suitable for the survival of the sperm in vitro. Each worm was cut with a disposable syringe needle at the oesophageal region allowing 56
the reproductive system to extrude. The undamaged reproductive
tract was1then carefully cut at the required region i.e. in the
middle of the uterus, at the seminal receptacle, or at the seminal
vesicle. A small cover-slip was then put over the saline drop and
the worm was examined under the microscope and photographed.
Some specimens were fixed with 2% gluteraldehyde (V/V made
in the saline solution) which was added dropwise to one side of the
cover-slip and was then sucked under from the other side using a
piece of blotting paper.
II.E.3. Observations
Mature sperm in this species were seen to be amoeboid and
non-flagellate. They had a broad, clear anterior region which was
the only region capable of producing pseudopodia and a rounded,
dense posterior region containing a nucleus, mitochondria, and
granular cytoplasm. There was no refringent body in these sperm
unlike the sperm of some other nematode species such as
A. lumbricoides (Foor, 1970).
The sperm in the seminal vesicle of the male did not produce
pseudopodia and there was no sign of any other type of sperm movement.
The sperm were irregularly rounded and did not show any enteric-
. posterior differentiation, granular cytoplasm filling the whole sperm
(Fig. 12). When the sperm were in the seminal vesicle of the male
they were not distinct and appeared as a granular mass, but when they
were released into saline solution they became clearly defined as
irregularly rounded cells. 57
••
Fig. 12 Undifferentiated sperm of seminal vesicle of male P. redivivus (x 2500).
Fig. 13 Beginning of differentiation of sperm from the uterus of the female P. redivivus into a clear anterior and a granular posterior portion while they are still in jelly like semen (x 2500). 58
The rounded sperm of the seminal vesicle of the male became
amoeboid when they were released into the uterus at insemination but
sperm which went into the post-vulvar uterine sac took up to 30 minutes
to become amoeboid. During insemination the sperm were transferred
into the uterus in the form of a mass in the jelly-like semen (Fig. 13).
After a few minutes the semen disappeared and the sperm became
differentiated into a clear anterior and a granular posterior portion
(Fig. 14). The sperm produced pseudopodia and arranged themselves in
chains, joining together anterio-posteriorly (Fig. 15). The chain of
sperm then moved anteriorly with the help of their pseudopodia.
However, sperm released into the posterior sac did not form chains
and remained scattered (Fig. 16) although they did eventually become
differentiated (Fig. 17). Normally only one chain was formed, composed
of 15 - 30 sperm, but if more than 30 sperm were present 2-3 chains
were sometimes formed.
Newly released sperm produced numerous pseudopodia over the
whole of their free surface in the chain (Fig. 15). With time the
number and size of the pseudopodia decreased, as did the activity of
the pseudopodia. The sperm were generally stored in the anterior
part of the uterus and after about eight hours the sperm no longer
produced pseudopodia and became rounded (Figs. 18 and 19), with a
clear anterior and a granular posterior end although they still
remained attached to each other in the chain. However, as the
sperm chain moved into the seminal receptacle for fertilization of the
oocytes (Fig. 20) the 7 - 8 anterior most sperm reformed pseudopodia,
the anterior-most sperm producing the largest and most numerous i'sei-al-0\)oci-+cL 59
Fig. 14 Fully differentiated sperm of P. redivivus from the uterus of the female (x 2500).
Fig. 15 Newly released sperm after differentiation and chain formation from the uterus of the female P. redivivus (x 2500). 60
Fig. 16 Newly released sperm in the post-vulvar uterine sac of female P. redivivus (x 2500).
Fig. 17 Sperm from the post-vulvar uterine sac of female P. redivivus after full differentiation (x 2500). 61
Fig. 18 Rounded sperm from the anterior part of the uterus of the female P. redivivus (x 2500).
Fit. 19 Scanning electron micrograph of sperm from the uterus of the female P. redivivus (x 3000). 62
Fig. 20 In utero sperm of P. redivivus entering seminal receptacle for fertilization (x 700). 63
(Figs. 21 and 22). The remaining sperm in the chain did not produce
pseudopodia until some of the anterior-most sperm were consumed
during fertilization.
The anterior-most pseudopodium of each sperm in the chain
acted as a holdfast with the posterior region of the next sperm. From
the lateral sides the sperm produced motile pseudopodia (Figs. 21 and
22) which moved posteriorly down the sperm where they disappeared,
enabling the chain to move in a forward direction. These pseudopodia
were capable of applying considerable force against the walls of the
uterus or against any other object in it and they were seen to push
eggs posteriorly down the uterus and to move into the seminal receptacle against the contraction of the sphincter muscle at the
utero-spermathecal junction.
When living sperm producing pseudopodia were fixed in a
small amount of 2% gluteraldehyde the sperm stopped producing
pseudopodia and became rounded. A few minutes later the anterior
sides of the sperm started to shrink producing a characteristic
wrinkled appearance (Fig. 23). These wrinkles looked very much
like pseudopodia and were found in the same region of the sperm,
there was therefore every chance of mistaking these wrinkles for
pseudopodia. 64
Figs. 21 & 22 Sperm from the seminal receptacle of the female P. redivivus prior to fertilization showing variation in appearance (x 2500). 65
Fig. 23 Appearance of sperm from the seminal receptacle of the female P. redivivus after fixation in gluteraldehyde at room temperature (x2500) 66
SECTION III
Copulatory behaviour of male P. redivivus
III.A. Post-moulting copulatory behaviour of adult male P. redivivus
III.A.1. Experimental procedure
A fourth stage, moulting male was placed in the centre of an agar plate together with E. coli and four newly moulted virgin females.
The male was continuously observed under the microscope during its final moult and its post-moulting behaviour recorded. The experiment was repeated three times.
III.A.2. Results
Immediately after moulting male P. redivivus fed intensively for 15 - 20 minutes during which time the male was practically immobile and did not respond towards female worms. After this short feeding period the male showed normal locomotory activity but still did not coil around female worms, even if they werqfeeding alongside the male. After about an hour (from moulting) the male began to show some response towards females and whenever the male came in contact with a female coiling occurred. However, this coiling activity was of a very short duration and the male soon recommenced feeding. This change in the male's behaviour was 67
concurrent with changes occurring in its reproductive tract, sperm
being stored in the seminal vesicle within one hour of moulting and
bubble-like structures appearing in the posterior region of the vas deferens (Section II.A.). As the post-moulting period lengthened the duration and frequency of male coiling increased.
There were often several attempts by a male to coil around a number of females before the male copulated. The first copulation of the male occurred between one and two hours of moulting depending
mainly upon the temperature, the amount of food available and the density of female worms. The whole process of copulation was completed within one minute. During the first copulation the male released about 20 - 30 sperm together with fluid from the vas deferens and the bubble-like structures from the posterior vas deferens
(Section II.D.). After the first copulation the male fed for most of the time and its response towards female worms was reduced to a minimum; it did not coil around another female even if its posterior end touched the female. Almost the same behavioural sequence followed' as described for the period between moulting and first copulation before the male copulated for a second time. The duration of the second copulation and the number of sperm transferred did not vary significantly from the first copulation. The intervals between the second, third and fourth copulations were each approximately 2 hours, this was reasonably similar to that between the final moult and the first copulation. The number of sperm transferred remained at about
20 - 30 per copulation. • 68
III.B. The rate of copulation, the number of sperm transferred
and the life span of normally copulating males.
III.B.1. Experimental procedure
A fourth stage, moulting male was placed with eight newly
moulted, virgin females on an agar plate containing E. coli. At
12 hour intervals the number of females which had copulated and the number of sperm and fertilized eggs present in each female was recorded.
After each 12 hour period the male was transferred to another plate containing a fresh batch of newly moulted, virgin females and E. coli.
During the day the copulatory activity of the male was also observed under the microscope. Each male was observed until it died. A total of five males were studied.
III.B.2. Results
The results of this experiment are shown in Figs. 24 and 25.
The results clearly showed that the rate of copulation was closely related to the rate of sperm transferred in each 24 hour period. The mean number of copulations per day varied between 8.4 and 10.8 over an
8 day period from moulting with a mean of 204 to 366 sperm transferred per day. This finding was in agreement with the observation made in the previous section (III.A.1.) that the male copulated every 2 — 3 hours for the first few copulations and suggested that the male continued to copulate at this rate for most of its life. However, the mean number -15
>. -12 o >. 360 9 .1(
cn z z 0 240 -6 'I= 2 cc w ct. 0 120 -3 ()
0 3 4 5 6 7 8 9 10 DAYS AFTER FINAL MOULT
Fig. 24 Rate of copulation and number of sperm transferred during the adult life of male P. redivivus. (3), copulations per day; (o), number of sperm transferred per day. 40 -
>-
CL 30 0 o cc 20 LiJu_
10 2 w
0 1 2 3 4 5 6 7 8 9 10 DAYS AFTER FINAL MOULT
Fig. 25 Mean number of sperm transferred/copulation/day during the adult life of male P. redivivus. Copulation, COP.
O 71
of sperm transferred per copulation per 24 hours varied from 22 to 34 over
this 8 day period; this was slightly greater than during the early
copulations. The data shown in Fig. 25 suggested that the mean number of
sperm transferred per copulation per 24 hours tended to increase with
the age of the male until the 6 - 7th day after which it declined.
The life span of males allowed to copulate ad libidum varied
from 9 - 10 days and during their life they copulated 71 - 101 times
and released 2093 - 2922 sperm. After 8 days, during the last few
days•of the male's life the rate of copulation declined considerably
and finally the male stopped feeding, became inactive and eventually
died. This final inactive stage lasted about 30 - 40 hours and during
this period the male became considerably smaller with the testis much
reduced in size and the vas deferens distorted. However, the seminal
vesicle was generally still full of sperm at the time of death.
III.C. Development and life span of virgin males
III.C.1. Experimental procedure
A fourth stage larval male was placed with E. coli in the
centre of an agar plate and its development and behaviour noted at
12 hour intervals from the final moult for four days. The male
was transferred to a fresh agar plate every 24 hours. A total of
•five males were studied.
To examine the mortality rate of virgin.males,about 70 fourth-
stage larval males were separated from the main culture and allowed to 72
• moult. After 12 hours a batch of 50 males was separated and kept at 25°C; the nematodes were transferred to fresh agar plates every
24 hours and during each transfer the number of dead worms was recorded. This experiment was repeated with 3 more batches of males.
III.C.2. Results
It was apparent that males prevented from copulating continued with the normal physiological maturation of the reproductive system.
After the final moult the virgin male fed and moved normally and its sperm were stored in the seminal vesicle. Twelve hours after moulting the seminal vesicle was granular in appearance due to the storage of
large numbers of sperm, and the band of 7 - 8 clear cells (Section II.A.) appeared around the seminal vesicle. As the seminal vesicle filled with sperm this band of cells appeared to move anteriorly. After one or two days, isolated virgin males had a compact mass of sperm in the seminal vesicle which in some individuals extended posteriorly into the anterior region of the vas deferens. The cells of the vas deferens became filled with fluid and the bubble-like structures in the posterior region of the vas deferens reached their maximum size within 12 hours of moulting. In
old virgin males, and in long isolated males, these bubble-like structures
varied considerably in size.
During the physiological maturation of the male reproductive
tract some behavioural changes were observed. In the first 12 hours
after moulting males spent most of their time feeding and in normal
locomotory activity, the latter increasing with the interval of 73
isolation. After 12 hours isolated males showed a decline in both feeding and loncomotory activity. During longer isolation periods of several days some males coiled around their own bodies (Fig. 26) and remained in this position for several minutes.
The mortality rate of virgin males is shown in Fig. 27.
Mortality increased rapidly, there being 24% mortality within 24 hours of moulting and over 99% mortality within 6 days.
M.D. Effect of short isolation periods on the copulatory
behaviour of male P. redivivus
III.D.1. Experimental procedure
Fourth stage, moulting males were kept individually on agar plates at 25°C and the exact time of moulting recorded. After
6, 12, 18, 24 or 30 hours from moulting, an isolated male was placed with five newly moulted females and its copulatory activity observed.
The duration of pre-insemination coiling, the number of sperm transferred, and the duration of post-insemination coiling and attachment were recorded. At least four males were examined for each period of isolation.
III.D.2. Results
As described earlier (Section III.C.2.), if a male did not copulate within 2- 3 hours of its final moult or of a previous copulation, 74
Fig. 26 Coiling of a long isolated male P. redivivus around its own body. 75
0 1 2 • 3 4 6 DAYS AFTER FINAL MOULT
Fig. 27 Mortality rate of virgin male P. redivivus. 76
sperm production and other physiological changes in the male's reproductive tract continued and these changes seemed to affect both the general behaviour and the copulatory behaviour of the nematodes.- Short isolation_ of. the male from a female affected its copulatory behaviour in the following way:
III.D.2a. The duration of pre-insemination coiling:
The results are shown in Fig. 28a. After 6 hours of separation male P. redivivus took an average of 4.3 - 2.6 (S.D.) minutes to locate the vulval opening after coiling commenced. This location time increased as the separation period increased. After
12 hours separation males took 5.6 1: 3.4 minutes to locate the vulva and after 24 and 30 hours location took on average 7.0 - 4.9 minutes.
Although the duration of location period appeared to increase this was not found to be significant (P = > 0.1). An increased location time was observed in some cases to be due to the male probing the dorsal side of the female for a considerable period and in others to the failure of the male to insert its spicules into the vagina immediately the vulva was touched.
III.D.2b. The number of sperm transferred per copulation:
The results are shown in Fig. 28b; they clearly indicate a steady increase in the number of sperm transferred at each copulation up to 12 hours of separation, 2 - 3 hour separated males transferred 77
14
13
12
11
10
w 2 I- 6- 5 - NG 4 OI LI C 3
2
1
0 6 12 18 24 30
ISOLATION PERIOD ( HOURS
Fig. 28.a. The effect of short separation from females on the duration of pre-insemination coiling • of male P. redivivus. Fitted regression line y = 4.45 + 0.08x. NUMBERO FS PERM T RANSFERRED 100 - 120 - 130 - 110 - 140 - 20 -120 80 - 50 •300 30 180 90 - 10 -60 60 -360 40 -240 70 - 0
Fig. 28.b.Theeffectofshortseparation fromfemalesonthenumber
COI LI NGTI ME ( SECO NDS ) coiling ofmaleP.redivivus. of spermtransferredanddurationpost-insemination transferred; (o),durationofpost-insemination coiling. 6
ISOLATION 12
PERIOD (HOURS)
18 (o), numberofsperm
24
30 78 79
20 - 30 sperm at the time of copulation, whife 12 hour separated males transferred 82 - 113 sperm. After 12 hours separation, however, the mean number of sperm transferred remained fairly constant although there was considerable variation between individual males (see Appendix).
III.D.2c. The duration of post-insemination coiling and attachment:
The results are shown in Fig. 28b. Normally the male copulated within 2 - 3 hours of moulting, and immediately after insemination they withdrew their spicules, uncoiled their tail and
Table 2
Effect of short separation of male
P. redivivus from females on post-insemination attachment
Hours Duration of post-insemination attachment (sec.)
males
separated Replicates 1 2 3 4 5
6 0 0 0 0 0
12 0 0 3 0 68
18 0 0 .85 30 10
30 5 7 0 9 125 80
moved away from the female (Section II.D.5.). However, as the interval between moulting and the first copulation increased, the male tended to prolong its post-insemination coiling. For example after 6 hours separation males coiled around the female for
60 - 80 seconds, while after 24 hours separation coiling lasted between 240 - 360 seconds.
Post-insemination attachment of the male to the female also appeared to increase with the separation period (Table 2). No post-insemination attachment was observed with 6 hour separated males, but 50% of the males were seen to be attached after
12 hours isolation; this increased to 80% with 30 hour isolated males.
III.E. Effect of long isolation period on the libido of
male P. redivivus
III.E.l. Experimental procedure
Two hundred fourth-stage larval males were allowed to moult on agar plates with E. coli at 25°C. After 12 hours the moulted males were replaced with freshly grown E. coli in the centre of an agar plate for ageing at 25°C. At 24 hour intervals ten males were each transferred to a fresh agar plate and each male kept with five newly moulted, virgin females for one hour. The number of females which copulated was recorded and the copulated females were separated for 24 hours at 25°C for fertilization of their oocytes to occur; the number of eggs fertilized was then recorded. This experiment was repeated three times. 81
III.E.2. Results
The results are shown. in Fig. 28c. As in the case of the life span of isolated males (Section III.C.2.) the libido of long- isolated, virgin males declined significantly (P =< 0.001). After
24 hours isolation 73% of the males were able to copulate, whereas only 33% copulated after 96 hours isolation.
In fact, the one hour test period, for the assessment of libido (copulatory activity) in male P. rediVivus was selected, because.trial experiments showed that the frequency and duration of pre-insemination coiling was much reduced for isolated males. It was observed that isolated males with reduced bubble-like structures in the vas deferens did not copulate until they had developed fully.
Normally, the storage of sperm in the seminal vesicle and the increase in the size of these bubble-like structures took place in synchrony after copulation.
The males which copulated transferred 60 - 150, apparently normal, sperm. In a few instances the sperm also went into the post-vulvar uterine sac and in a very few cases no sperm were transferred at copulation. At 24 hours after copulation 15 - 26 eggs were fertilized by the sperm (Fig. 28c). There was no significant change in the number of oocytes fertilized with increasing isolation (P = > 0.1) showing that the sperm did not lose their capacity to fertilize oocytes with age when stored in the seminal vesicle of the male. In most cases sperm were present in the seminal receptacle of the female 24 hours after insemination.
82
— 28
— 24 0 UJ :::* 100 20
l- c) 80 —16
Oc 60 —12 0
L1J
Li) 0 liJ n-20 — 0 24 48 72 96 HOURS ISOLATED AFTER FINAL MOULT Fig. 28.c. The effect of long isolation periods for virgin male P. redivivus on the percentage of males copulating in a one hour period and on the number of oocytes fertilized by their sperm within 24 hours of copulation. (o), percentage male copulated; (a), number of oocytes fertilized. Fitted regression lines y = 90 - 0.63x and y = 19.97 - 0.01. 83 SECTION IV Development and life span in female P. redivivus IV.A.1. Introduction Preliminary observations on the final moult and first copulation in P. redivivus showed that if the female copulated immediately after moulting the first egg passed into the uterus within hours of copulation and that further eggs appeared at approximately hourly intervals. However, if copulation was delayed for 3 - 4 hours the initial rate of egg production was increased, and if copulation did not occur within 24 hours unfertilized oocytes appeared in the uterus. The following experiments were designed to further investigate development in the reproductive tract of virgin females and to compare it with that in normally copulating females. IV.A.2. Experimental procedure Fourth stage females were placed in individual petri dishes containing agar with E. coli (Section I.D.) and the plates incubated at 25°C. The development of the ovary, oviduct, seminal receptacle, uterus and post-vulvar uterine sac in each virgin female was observed (at X 40) at- the time of final moult (duration four hours), and subsequent observations were made at 24 hour intervals from the completion of the moult until death. The nematodes were transferred 84 to fresh agar plates every 24 hours. The development of normally copulating females was also studied at 24 hour intervals. To compare the rate of mortality in virgin and copulating females, fourth stage females were isolated on agar plates with E. coli and after 12 hours moulted females were transferred to ten fresh agar plates with E. coli (100 females/plate). Fifty males were added per plate to five plates to ensure that all the females had copulated. The plates were then kept in an incubator at 25°C, and the female nematodes were transferred to fresh agar plates every 24 hours, and in the case of plates with copulated females the males were replaced by 50,fresh individuals. The number of virgin and copulated females alive was recorded at each transfer i.e. at 24 hour intervals. IV.A. . Results IV.A.3a. Development in virgin females: At the final moult females usually had 3 - 4 oocytes in the oviduct, while the uterus was clear and without any oocytes. The seminal receptacle was thin and transverse to the intestine, the ovary was small, clear and somewhere in the vulval region lateral to the intestine. The post-vulvar uterine sac was also clear and empty. Twenty four hours after the final moult the ovary had already produced 20 - 25 mature oocytes which were stored principally in the oviduct; as a result the ovary was displaced posteriorly. Three to 85 four oocytes had also been passed into the uterus via the seminal receptacle (Fig. 29a). After 48 hours the number of oocytes in the oviduct had increased to about 30 and to accomodate this number of oocytes the oviduct was distended and looped (Fig. 29b); about 10 - 15 oocytes had passed into the uterus by this time. The female had also grown considerably in size, both in length and diameter and the seminal receptacle had moved ventrally towards the uterus because the oviduct was enlarging faster than the uterus and in some females the seminal receptacle was almost parallel to the intestine. Seventy two hours after moulting many more oocytes had passed into the uterus (Fig$.30a, 32), and the post-vulvar uterine sac had started to develop a small ridge in its lumen. By the fifth day the virgin female had the maximum number of oocytes in the oviduct and uterus (Figs. 30b, 31 and 32). From this time the process of degeneration started. The number of oocytes in the oviduct decreased because of the failure of the ovary to replace oocytes passed into the uterus and the degeneration and possible reabsorption of oocytes by the oviduct. The oocytes in the uterus also began to degenerate and eventually the uterus became filled with fluid. As the process of degeneration of the oocytes started in the uterus a dense drop of proteinaceous or fatty material appeared at its distal end near the vaginal opening. This dense material appeared to pass slowly into the post-vulvar uterine sac where it filled up the whole sac (it is possible that material may also have been secreted by the post-vulvar 86 Fig. 29 Reproductive morphology of virgin female P. redivivus at one and two days after moulting. A, one day old female; B, two days old female. Ovary, OV; oviduct, OVD; post-vulvar uterine sac, PVUS; seminal receptacle, SEM RECP; uterus, UT. 87 88 Fig. 30 Reproductive morphology of virgin female P. redivivus at three and five days after moulting. A, three day old female; II, five day old female. Oviduct, OVD; post-vulvar uterine sac, PVUS; seminal receptacle, SEM RECP; uterus, UT. 89 50 - 0 1 2 3 4 5 6 7 8 9 10 DAYS AFTER FIN AL MOULT Fig. 31 Storage of oocytes in the oviduct of copulated (o) and virgin female (0) P. redivivus with age. 50 cn 40 — 30 cc Lc 20 0 cn U.1 >0 1 0 0 1 2 3 4 5 6 7 8 9 10 DAYS AFTER FINAL MOULT Fig. 32 Storage of oocytes or larvae in the uterus of copulated (o) and virgin female (o) P. redivivus with age. 92 uterine sac itself). The function, if any, of this material in copulated or uncopulated female was unknown. Most virgin females died within ten days of moulting and the remaining females had only a few vestigeal oocytes remaining in the oviduct, while almost all the oocytes had dissolved in the uterus and the post—vulvar uterine sac was full of dense material. IV.A.3b. Development in copulated females: The most marked difference between copulated and virgin females was in the rate of extension of the uterus in the early stages of adult development; the uterus rapidly became larger than the oviduct in copulated females (Fig. 3) but remained smaller than the oviduct in virgin females (Fig. 29). This was due to the passage of most oocytes from the oviduct into the uterus for fertilization. One day old adult copulated females had the largest number of embryos and larvae in the uterus, thereafter the number declined rapidly (Fig. 32). A similar trend was found for the oocytes in the oviduct of copulated females (Fig. 31). In virgin females the rate of passage of oocytes into the uterus was much slower and the maximum number of oocytes in both the oviduct and uterus was reached at the fifth day after the final moult (Figs. 31 and 32). As the uterus increased in size faster than the oviduct in the copulated female the seminal receptacle remained transverse to the intestine (Fig. 3). This was in contrast to the virgin female where the seminal receptacle was parallel to the intestine due to the greater 93 rate of growth of the oviduct (Figs. 29 and 30). The other principal difference between copulated and virgin female was in the development of the post-vulvar uterine sac. In copulated females it functioned as a store for excess fluid from the vas deferens. This fluid later probably passed into the uterus or was absorbed via the wall of the sac into the pseudocoelom. Sometimes it also served to store excess sperm which could not be accomodated in the uterus at the time of copulation and which later passed into the uterus. In older virgin females the post-vulvar uterine sac filled with dense material as described above. IV.A.3c. Mortality of virgin and copulated females: Both copulated and virgin females had approximately asymmetric sigmoid mortality curves (Fig. 33). Mortality increased rapidly in both copulated and virgin females until 6 - 7 days after the final moult, after which the mortality rate was somewhat greater in the copulated females than in the virgin females (Fig. 33). There being a 100% mortality in copulated females after 11 days, while a few virgin females lived over 20 days. Ageing copulated females stopped feeding, decreased in size and eventually ceased to move and died. However, in some cases mortality occurred among apparently healthy virgin females of normal size, with large number of oocytes, and with food in the intestine. This phenomenon was more apparent in young populations of virgin females than in older ones. 0 1 2 4 5 6 7 8 9 10 11 12 13. 14 15 16 17 18 19 20 DAYS AFTER FINAL MOULT Fig. 33 Mortality rate of virgin female P. redivivus compared with normally copulating females. (o), for virgin females; (o), for copulated females. 95 SECTION V Sex attraction in P. redivivus V.A. Introduction There has been little work on the degree of sex attraction in virgin and copulated female nematodes, or in newly moulted or gravid females. In the present experiments sex attraction in P. redivivus has been examined, the possible attractiveness of female P. redivivus to the male being the principal interest. The female produces 80 - 90 eggs during its life and only copulates 4 - 5 times which suggests that any attractiveness could vary during the life span; whereas the male produces 2000 - 3000 sperm and copulates on average every 2 - 3 hours and may be responsive at all the times towards females. V.B. Test apparatus for sex attraction experiments and experimental procedure. Water agar (1.5% W/V) was poured into 50 X 15 mm plastic petri dishes to a depth of 5 mm. After solidification of the agar, two 10 mm wide, migration strips were made in a V - shape by selective removal of agar, so that the apex of the V formed the inoculation zone and the ends formed the two test zones. Parallel lines were etched 96 across the bottom of the petri dish to divide each agar strip into three sections (Fig. 34). Test chambers were made by cutting 6 - 7 mm long tubular pieces from a 4 mm diameter plastic drinking straw care being taken that the cut ends of the straws were smooth and flat. Adhesive (UHU glue, Fismar Ltd., Eire) was applied to the rim of one end of the piece of straw and this was then fixed onto the centre of a 5 mm diameter disc of Whatmann No.1 filter paper. After allowing the adhesive to set the joint between the straw and the filter paper was checked under the dissecting microscope to ensure that there were no spaces for the worms to escape. The centre of the filter paper had also to be free of adhesive to allow any attractant to pass freely into the agar. . A large drop of water agar (2% W/V) was placed at the end of one of the open arms of the V - shaped strips, and the straw with the filter paper side facing down was pressed into the moulten agar until the filter paper almost touched the solid strip and the moulten agar had covered the edges of the filter paper and the lower part of the straw tube. A similar straw was attached to the other arm of the V - shaped strip (Fig.34). After solidification of the water agar surrounding the lower part of the straws a small drop of agar was placed inside each straw. Care was taken that the agar drop did not stick to the sides of the straw and that it covered the whole surface of the filter paper. The thickness of the agar inside the straw was not allowed to exceed 2 - 3 mm. After solidification of the agar inside the straws (now called test and control chambers) an equal 97 A B Fig. 34 Apparatus used for sex attraction experiments. A, piece of plastic drinking straw fixed on filter paper; B., straw in a moulten agar drop; C, small drop of moulten agar inside straw; D, E. coli added into the straw now called "chamb"Fil E, the whole apparatus with two chambers at open ends and inoculation zone at apex of V. 98 amount of freshly grown E. coli was added to the centre of each chamber without contaminating the walls. A group of 100 worms to be tested for the production of sex attractant was placed on the E. coli in one of the chambers (to be called the test chamber). The control chamber contained only E. coll. The petri dish was then covered and placed in an incubator at 25°C to allow time for any attractant to diffuse from the test chamber. After 24 hours 100 worms to be tested for their response to any sex attractant were placed in the inoculation zone at the apex of the agar V. After inoculation the plate was kept in the incubator for a further 6 hours and the number of worms in each of the three sections were counted. Each experiment was repeated ten times alternating the arms of the V used for the test and control chambers. The results were analysed statistically by chi-square and student's t-test. • Only the chi-square test results are shown in the text. V. C. Results V.C.1. Response of virgin females and adult males towards E. coli The main purpose of this experiment was to see if E. coli can influence the•distribution of the worms, and thus possibly affect the results of the sex attraction experiments due to an inbalance in the test and control chambers. The results are shown in Fig. 35. No significant difference in the number of worms migrating up each arm 50 - 50 A C 40 - z Z 40 0 0 30 - 30 U 0 (n 20 - I cn 20 w10 - w 10 J 2 2 9 A A L6- 50 - 50 B 0 re 40 w 40 LIJ LIJ co c 2 30 2 30 Z 20 20- 10 - 10 - section -2 +1 +2 +3 section -3 -2 -1 0 +1 +3 Fig. 35 Distribution of P. redivivus on.agar strips after 6 hours at 25°C. A, males in response to males; B, males in response to E. coli; C, virgin females in response to virgin females; D, virgin females in response to E. coli. A indicates where 100 respondant worms were added and c3'' or 9 indicates the position and sex of 100 attractant worms. 100 of the agar V was found ( P = > 0.05), indicating that E. coli did not interfere in the sex. attraction experiments. In fact greater numbers of worms remained in the inoculation zone than in the other experiments (Figs. 36, 37 and 38). V.C.2. Homosexual attraction and response of virgin females and adult males No significant attraction of virgin females towards other females was found (Fig. 35, P = > 0.995), nor between males (Fig.35, P = > 0.995). In both of these experiments, in fact, most of the worms remained in the inoculation zone. V.C.3. Attraction and response of fourth stage larval females tO adult males The results for fourth stage larval females are presented in Fig. 36. No significant difference between the number of males which migrated towards the test and control chambers was found (P = > 0.995). About two thirds of male P. redivivus migrated out of the inoculation zone, one third towards the test chamber and one third towards the control chamber. Similarly, there was no significant response of fourth stage larval females towards adult males (P = > 0.05). In contrast, in experiments in which more than half of the females had moulted to the adult stage or were in the process of O (.9.50 A <30 - 0 20 CC 111 °3 1 0 2 z 9 T A A o 50 - D F Cl) U) `2.51 30 X20 - 0cd io- I I cc, r a -I I 2 A A -2 -1 0 +1 +2 +3 Z section -3 2 -1 0 +1 +2 +3 -3 -2 -1 0 +1 +2 +3 Fis..36 Distribution of P. redivivus on agar strips after 6 hours at 25°C. A, males in response to fourth stage females; B, fourth stage females in response to males; C, males in response to newly copulated females; D, newly copulated females in response to males; E, males in response to gravid females; F, gravid females in response to males. A indicates where 100 respondant worms were added and d and 9 indicates the position and sex of 100 attractant worms. 102 moulting by the end of the test period, a.significantly greater number of males migrated towards the test chamber than towards the control chamber (P = < 0.001, see Appendix). •V.C.4. Attraction and response of normally copulating females to adult males No significant attraction of males towards recently copulated females (Fig. 36, P = > 0.05) or gravid females (Fig. 36, P = > 0.995) was found. Similarly, no significant attraction of newly copulated females (Fig. 36, P = > 0.995) or gravid females (Fig. 36, P = > 0.995) towards males was found. V.C.5. Attraction and response of ageing virgin females to adult males P. redivivus Females became attractive to males for the first time after their final moult ( P = < 0.001), however, as the virgin females grew older they slowly lost their attractiveness (Fig. 37 and 39a) and 192 hour old virgin females did not attract significantly higher number of males than the control chamber (P = > 0.995). Newly moulted virgin females were also significantly respondent towards adult males (P = < 0.001) and remained respondent throughout their whole adult life (Figs. 38 and 39b) (P = < 0.001 for 96 and 240 hour old virgin females). 50 A B C 40 z o 30 — 9 A A section -3 -2 0 +1 +2 +3 050 D E ff, 4 0 2 30 20 10 IT A I I A section -3 -2 -1 0 +1 +2 +3 0 +1 +2 +3 Fig. 37 Distribution of male P. redivivus on agar strips after 6 hours at 25°C in response to virgin females of varying age. A, newly moulted females; B, 48 hour old virgin females; C, 96 hour old virgin females; D, 144 hour old virgin females; E,.192 hour old virgin females. A indicates where 100 respondant males were added and indicates the position of 100 attractant females. 50 B 40 z o30 w 20 Cf) ••••■ X 10 w I I. a 2 A UJ ;50 D E F 0 ce 40 - 30 z 20 10 1 I section -3 -2 -1 0 +1 +2 +3 -3 -2 -1 0 +1 +2 +3 -3 ÷1 +2 Fig. 38 Distribution of virgin female P. redivivus of varying age on agar strips after 6 hours at 25°C in response to males. A, newly moulted females; B, 48 hour old virgin females; C, 96 hour old virgin females; D, 144 hour old virgin females; E, 192 hour old virgin females; F, 240 hour old virgin females. A indicates where 100 respondant females were added and d indicates the position of the 100 attractant males. 100 90 80 70 0 60 2 50 U.1 0 *5f 40 z LU rc.. 30 LU °- 20 10 0 2 4 6 8 • 0 2 4 6 8 10 AGE (DAYS) OF VIRGIN FEMALES Fig. 39.a. Percentage motility (o) and Fig. 39.b. Percentage motility (o) and response (11) response (0 of male of virgin females of increasing age P. redivivus towards virgin towards males. females of increasing age. 106 Figs. 39a, b show that the % motility of males and virgin. females had no obvious relationship with the attractiveness or responsiveness of the virgin females. They show for example that the % motility of ageing virgin females steadily dropped after 96 hours from the final moult, although there was still a significant response of virgin females towards adult males. Also, although virgin females slowly lost their attractiveness for adult males as they aged they did not lose their responsiveness towards adult males (Figs. 39a, b). 107 SECTION VI Copulatory behaviour of female P. redivivus. VI.A. Timing of first copulation in the female VI.A.1. Introduction Preliminary observations at room temperature showed that immediately after moulting the adult female fed for a period of 15 - 20 minutes and during this period the nematode was practically immobile and did not respond towards the male. Newly moulted females were not observed to copulate; this suggested that post-moulting changes were necessary before the female is receptive to the male, although the time between moulting and the first copulation was very variable. It was possible for example that secretions from the female reproductive organs, such as the ovary or uterus, were required to stimulate the male to tran fer its sperm. To investigate this further, the time taken for newly moulted females to copulate in the presence of varying numbers of males was recorded. VI.A.2. Experimental procedure Moulting females were placed on to individual agar plates with E. coli and to each plate were added one, five, ten or fifteen males which had been isolated from females for 3 - 4 hours. Final 108 moulting and the first copulation of the female was observed under the microscope and the time interval between the two events recorded. These experiments were conducted at 25°C and were repeated five times for each group of males. VI.A.3. Results During the initial stages of moulting by the female the males continued to feed in its vicinity without apparent awareness of the female's presence. Occasionally a male would be seen to coil briefly around the female and then move away. However, as soon as'the female began to undergo ecdysis the males were attracted and most of them gathered around the female. Thus the female appeared to become attractive to the male during ecdysis. It was observed that the males continuously attempted to copulate after the female's ecdysis, the time taken for the first copulation to occur depending upon the number of males present in the vicinity of the female (Fig. 40). It was also found that if a female in the early stages of moulting (The whole moulting process takes approximately four hours) was used instead of a female which had nearly finished moulting, a greater number of males accumulated in the region of the female. This suggested that moulting females secreted an attractant. This difference between females at various stages of moulting probably accounted for some of the variability in the time taken for the first copulation particularly when small numbers of males were used (Fig. 40). When 7 - 8 males were in the vicinity of the female during ecdysis, copulation was observed immediately after 109 0 . 5 10 15 NUMBER OF MALES PRESENT Fig. 40 The effect of density of males on the timing of the first copulation of female P. redivivus. Fitted regression line y = 39.17 — 2.47x. 110 moulting or even in some cases before ecdysis had been fully completed. After the female had copulated the males remained coiled around the female for over 30 minutes. Sometimes two copulations took place within ten minutes of moulting when sufficient numbers of males were near a moulting female. In about 80% of the newly moulted females feeding stopped as insemination took place; preliminary studies showed that the interval during which the female did not feed varied from a few seconds to ten minutes. VI.B. The effect of ageing on the first copulation of the virgin female VI.B.1. Introduction Virgin females remained attractive to the adult male for most of,their,life possibly due: to the'presence of large numbers of oocytes-, in the oviduct (Section V.C.5.). In this experiment the effect of ageing on the ability of Virgin females to copulate was examined. VI.B.2. Experimental procedure Fourth stage larval females were put on to agar plates with E. coli for moulting at 25°C. After 12 hours, moulted females were separated and transferred to agar plates with fresh E. coli and aged at 25°C. As with previous experiments the nematodes were transferred to fresh agar plates every 24 hours. Batches of ten females were taken for copulation experiments at 48, 96, 144, 192, and 240 hours after 111 moulting. Each female of the batch was placed with five males which had been previously kept separated from females for 18 hours, and after one hour the number of females which had copulated was noted. In copulated females the number of eggs produced within the following 24 hours was also recorded. Each experiment was repeated three times. VI.B.3. Results Approximately 90% of the virgin females copulated within one hour and there was no significant change in copulation with age (P = > 0.1). However, the egg production showed a significant increase (P = < 0.001) from 48 hour to 192 hour and then dropped significantly again for females separated for 240 hours (P = < 0.001, Fig.41). These results showed that as .for• sex attraction the ability: of virgin females to copulate was not significantly affected by age. However, it was noticed that in a few females which did not feed (the reason for which was not known) and which contained few oocytes copulation did not usually take place. VI.C. Factors influencing the second copulation of female nematodes VI.C.l. Introduction Attempts to make generalizations about the second copulation in female P. redivivus are even more difficult than for the first PERCEN T AGE COPULAT ED 100 80 50 60 30 90 70 40 20 10 0 Fig. 41Theeffectof ageingofthevirginfemaleP.redivivus onthepercentageoffemales copulating (o)forthe firsttimeandnumberofeggsproduced (o)within24hours of copulation.Fitted regressionliney=96.33—0.03x 48 HOURS OLDAFTERFINALMOULT 96 144 192 240 113 copulation. In preliminary experiments females were observed continuously for 8 - 10 hours after their final moult in order to measure the time interval between the first and second copulations. During these observations some females were seen to copulate again immediately after the first copulation, while others did not copulate again for several hours. In some cases a male coiled around the female and its spicules were inserted into the vagina but insemination did not occur. This type of unsuccessful copulation may occur up to ten times before full copulation (with insemination) takes place. In other females insemination occurred but the sperm were released into the post-vulvar uterine sac where they often remained for a considerable period before moving towards the seminal receptacle (Section II.E.3.). The appearance of fertilized oocytes and the hatching of larvae in the uterus had no apparent effect on further copulation of the female. Obviously there are several interacting factors determining the time of the second copulation in this nematode; these may include the number and age of sperm present in the uterus of the female, the rate of oocyte production by the female and the copulatory activity of the male. In the following experiments an attempt was made to assess the influence of such factors on the copulatory activity of the female. 114 VI.C.2. Experimental procedure VI.C.2a. The influence of male copulatory activity.on the copulatory behaviour of once-copulated females: Newly moulted females following their first copulation were placed on individual agar plates with E. coli and kept at 25°C. At four hourly intervals for 24 hours one of these females was transferred to a fresh agar plate with ten males which had been previously separated from females for 2 - 3 hours. The number of females which copulated within 45 minutes was recorded, together with the number of eggs present in the uterus at that time. Ten females were examined for each time interval. The whole experiment was repeated three times. To see if long-isolated males could force females to copulate prematurely the above experiment was repeated with males previously isolated for 12 - 4 hours. The results were analysed statistically using the Friedman two-way analysis of variance by ranks. VI.C.2b. Number and ageing of sperm in the uterus: Newly moulted females were copulated with males previously isolated for 12 4 hours, thus receiving approximately 80 - 110 sperm. The second copulation of these females was tested as in Section VI.C.2a. with males previously isolated 12 4 hours, until 115 90% of the females had copulated. VI.C.2c. Number and ageing of oocytes in the female: Females at the fourth larval stage were isolated and after 12 hours the moulted females were separated and placed with E. coli on fresh agar plates for ageing at 25°C. After 48 or 96 hours, 100 females were copulated with males previously isolated for 2 - 3 hours. The second copulation of these females was tested as in Section VI.C.2a. with males previously isolated for 12 4 hours, until 90% of the females had copulated. The results were compared with newly moulted copulated females. VI.C.3. Results VI.C.3a. The influence of male copulatory activity on the copulatory behaviour of once-copulated females: The number of females copulating for a second time with increasing time of separation from the males is shown in Fig. 42. There was no significant difference in the first 12 hours between females exposed to males previously isolated for 3 - 4 hours or for 12 - 4 hours (P = > 0.1). However, in the next 12 hours a significantly, higher number of females copulated with males previously isolated for 12 4 hours than for 3 - 4 hours (P = < 0.05). 100- 90- 80- 70- 2 60- o 50- w 40- z tije) 30- w a. 20- 10 - 0 4 8 12 16 20 24 28 32 36 40 HOURS AFTER FIRST COPULATION Fig. 42 The effect of activity of the male and number of sperm received at the first copulation of the female P. redivivus on the % of the females copulating for the second time with increasing time intervals after the first copulation. (o), females with < 30 sperm and 2 — 3 hours isolated males; (4), femiles with < 30 sperm and 12 — 4 hours isolated males; (0), females with > 80 sperm and 12 — 4 hours isolated males. 117 About 15% of the females copulated while still possessing sperm from the previous copulation; these were mostly restricted to those tested in the first 12 hours from the first copulation in experiments with males previously isolated for 3 - 4 hours. With males previously isolated for 12 - 4 hours, females copulated with sperm still remaining from the previous copulation over a wider period from 4 - 24 hours (Fig. 43). VI.C.3b. The number and ageing of sperm in the uterus: The number of females inseminated with approximately 80 - 110 sperm during the first copulation (i.e. with sperm numbers greatly in excess of the number of oocytes that can be fertilized in 24 hours, Fig. 44) that Copulated for a second time with increasing time of separation from males is shown in Fig. 42. None of these females copulated within the first 8 hours of the previous copulation and very few females copulated within 16 hours. After 16 hours, however, there was a steady increase in the number of females which copulated, reaching 90% after 40 hours from the first copulation. In the first 20 hours 100% of females which copulated had sperm remaining in the uterus from the previous copulation; in the following 20 hours 76% of the females which copulated had sperm in the uterus (Fig. 43). The number of females which copulated after exhausting their sperm supply increased rapidly after 32 hour separation. Preliminary experiments showed that the number of oocytes fertilized did not increase directly in proportion to the number of sperm received. This 118 90 80 - . 70- 60 - w B -J a. 50 - O Li, 40- z 30 ... w •Ix w 0- 20_ 10 - 0 4 8 12 16 20 24 28 32 36 40 HOURS AFTER FIRST COPULATION Fig. 43 The % of female P. redivivus copulating for a second time with or without sperm from the first copulation at varying time intervals after the first copulation. A, In females which received low number of sperm at the first copulation and copulated with sperm (o), without sperm (o). B, In females which received high number of sperm at the first copulation and copulated with sperm ( • ), without sperm ( [3 ). 119 Fig. 44 The effect of the number of sperm received and the number of oocytes present in the oviduct and uterus at the time of copulation on the rate of fertilization of oocytes in'the female P. redivivus. (e) for newly moulted females inseminated with < 30 sperm; (o) for newly moulted females inseminated with > 80 sperm; (■) for 48 hour old virgin females inseminated with < 30 sperm; (o) for 96 hour old virgin females inseminated with < 30 sperm. 120 38 36 34 32 30 28 26 o 24 - LIJ N = 22. 20 LL 18 >-o 16- 14 12- 10 • 8 6- 0 4 8 12 16 20 24 HOURS AFTER FIRST COPULATION 121 suggested that the sperm had a limited functional life and where the number of sperm exceeded the number of oocytes available during a 48 hour period egg production remained constant. As no more sperm were found after 48 hours this implied that they had degenerated. VI.C.3c: The number and ageing of oocytes in the females: The results of this experiment are shown in Figs. 44, 45, and 46. In 48 hour old virgin females a significant proportion of the oocytes were fertilized within the first few hours from copulation; probably most of these oocytes were already in the uterus at the time of copulation (Fig. 44). The rate of fertilization reached an apparent peak between 0 - 4, 4 - 8, and 4 - 16 hours after copulation for 96 hour, 48 hour and newly moulted adult females, respectively (Fig. 45). The maximum rate of fertilization was greatest in the females separated for 48 and 96 hours from moulting and lowest in the newly moulted females. The rate of fertilization of oocytes in 48 hour and 96 hour old females was generally greater than that in the newly moulted females particularly during the first 8 hours after copulation (Fig. 44). The rate of copulation in females which had unfertilized oocytes in the oviduct and uterus (those separated for 48 and 96 hours) was generally higher than in newly moulted, copulated females over the 16 hour period from copulation (Fig. 46). 4 03 0 w 2 w LL cr ``1 0 0 :-;72 0 4 8 12 16 20 HOURS AFTER FIRST COPULATION Fig. 45 The effect of age of female P. redivivus at the first copulation on the rate of oocyte fertilization. (❑) after final moult, ( 0) after 48 hour virginity, (in) after 96 hour virginity. - 104 90 80. 1- 74 260' 0 LLI O < 40, U 30 0_ 21:1 10 0 4 8 12 16 20 24 28 HOURS AFTER FIRST COPULATION Fig. 46 The effect of the age of -female P. redivivus at the first copulation on the % of females copulating for the second time with increasing time intervals after thT first copulation. (e) Newly moulted females with < 30 sperm and 12 - 4 hour isolated males, (IN) 48 hour old virgin females with < 30 sperm and 12 - 4 hour isolated males, (❑) 96 hour old virgin females with < 30 sperm and 12 - 4 hour isolated males. 124 VI.D.. The third copulation of female P. redivivus VI.D.1. Experimental procedure Newly moulted, copulated females were placed with E. coli on agar plates for 20 hours at 25°C. The aged females were then kept with males previously isolated for 2 - 3 hours and those females which copulated were tested for a third copulation with males previously isolated for 12 ± 4 hours as described in Section VI.C.2a. VI.D.2. Results The results are shown in Fig. 47. The increase in the number of females copulating for a third time with increasing time from the second copulation was similar to that observed after the first copulation up to 20 hours from the previous copulation (Fig. 42). This was particularly true if the females copulating without sperm from the previous copulation were considered (Fig. 47). In general the number of females which copulated with sperm remaining in the uterus was higher at the third copulation than at the second copulation. The number of embryos or larvae in the uterus and the release of the larvae from the female seemed to have little or no effect on copulation in the female or on the movement of sperm up the uterus towards the seminal receptacle. 125 90 80 70 60 0 w n 50 O 40 w z30 w rx EL 20 10 0 4 8 12 16 20 24 HOURS AFTER SECOND COPULATION Fig. 47 The 7. of female P. redivivus copulating for third time with ■ ) or without sperm (❑) from the second copulation at increasing time intervals after the previous copulation; compared with similar situation at the second copulation with sperm (s), without sperm (o). 126 VI.E. Behaviour of sperm of P. redivivus in vitro in the presence of oocytes VI.E.1. Introduction Earlier experiments have suggested that unfertilized oocytes may influence the motility of sperm in the uterus of female P. redivivus (Sections II.E. and VI.C.). The present experiment was thus designed to examine the effect of unfertilized oocytes on sperm at different stages of development. VI.E.2. Experimental procedure Sperm from the seminal vesicle of male P. redivivus and from the uterus and post-vulvar uterine sac of the female were separately mixed with unfertilized oocytes obtained from the oviduct of the female in a drop of saline solution (Section II.E.2.). A small coverslip was then placed over the saline drop and the sperm observed under the microscope for pseudopodial production. VI.E.3. Results No change in sperm behaviour was observed when they were placed with unfertilized oocytes. The sperm from the post-vulvar uterine sac * did produce a few small pseudopodia but this also occurred in the absence of oocytes. 127 GENERAL DISCU'SSION Copulatory behaviour and the role of the spicules and caudal papillae during copulation. As was described earlier in the introduction copulatory behaviour in nematodes has been shown to vary considerably between species. In P. redivivus, as in P. rigidus (Greet, 1964), Cylindrocorpus spp. (Chin and Taylor, 1969) and X. diversicaudatum (Trudgill, 1976), the male was found to coil its tail around the vulval region of the female during copulation. This is in contrast to many other species of nematode where the male just holds the female with the bursa and does not coil at all around the female (Fisher, 1972; Chuang, 1962; Anderson and Darling, 1964; _Jones, 1966; .Somnerville and Weinstein, 1964). It seems to be generally true that these different types of copulatory behaviour are reflected in the organisation of the copulatory muscles. These are well developed in species where coiling takes place and are either absent or poorly developed in species where coiling does not occur. The degree of coiling may also be dependent upon the number of copulatory muscles in a particular species. The copulatory muscles have been found to vary in number from 3 - 4 in oxyurids to 40 - 50 in ascarids (Chitwood and Chitwood, 1950). The other major difference between nematode species appears to be in the duration of copulatory behaviour which varies from a few seconds to several hours. P. redivivus appears to be intermediate in 128 this respect, copulation taking from 1 - 6 minutes in the present studies. In fact, it seems that there is often more variability between individuals of the same species than between different species, for example in R. teres copulation lasts from a few minutes to a few hours (Chuang, 1962). The actual process of copulation in nematodes starts with the penetration of the spicules and finishes when the spicules are withdrawn after insemination has occurred. The greatest variation occurs at pre-insemination coiling, which varies from one to 17 minutes in P. redivivus and the duration of which depends presumably upon the excitement of the male, and at post-insemination attachment which may last for several hours in species which secrete cement-like material during copulation, such as A. avenae (Fisher, 1972). In P. redivivus, like P. rigidus (Greet, 1964) and . X. diversicaudatum (Trudgill, 1976), the male's spicules remain constantly in touch with fenale's body, unlike R. teres (Chuang, 1962), P. teres (Jones, 1966) and Cylindrocorpus spp. (Chin and Taylor, 1969) where the spicules are periodically projected to make contact with female's body. This type of probing behaviour suggests a sensory function for the spicules (e.g. Lee and Atkinson, 1976) and recent studies on the spicules of various species have in fact shown that they contain nerve process and in some species the spicules also have pores at their distal ends. McLaren (1976) suggested a chemosensory function for those spicules with pores and a mechanosensory one for those without such pores. The present light microscope studies on P. redivivus have shown the presence of such pores at the distal end 129 of each spicule (Fig. 5). A similar sensory function has been suggested for the caudal papillae in nematodes (McLaren, 1976), and the presence of 15 caudal papillae in male P. redivivus (Figs..6 and 7) would certainly suggest an important role for these papillae in the orientation of the male nematode for copulation. In P. redivivus the spicules were found to make a channel through which the sperm entered the female (Figs. 6 and 7) as in some other nematode species (McLaren, 1976). However, in P. redivivus this channel was formed by the membranous velum of each spicule, whereas in Heterodera spp. it was formed by the hard sclerotized parts of the spicules (Clark, Shepherd and Kempton, 1973). Functional maturation of sperm in nematodes It now seems well established that nematode sperm only become functionally mature in the uterus of the female. Although the factors controlling this maturation of the sperm are still not understood, it seems reasonable to assume that the sperm respond to some chemical stimulus in the female reproductive tract. Foor and McMahon (1973), in describing the role of the glandular vas deferens in the development of sperm in A. suum were of the opinion that this organ also played an important part in the development of the sperm. They found that when fluid from the vas deferens of A. suum was injected into the seminal vesicle some sperm became functionally mature, although they took considerably longer than in the uterus of the female. This suggested that secretions from the vas deferens which pass into the uterus with . 130 the sperm at insemination may be partially responsible for the . functional maturation of the sperm in the uterus. The present observations on P. redivivus, have shown that the sperm become differentiated into a clear cytoplasmic portion and a granular portion while in the jelly-like semen. This suggested that it was the secretions' from the male rather than from the female which stimulated differentiation of the sperm. However, the finding that sperm released into the post-vulvar uterine sac of female P. redivivus did not immediately differentiate suggested that the stimulus for structurally mature sperm to become mobile was only present in sufficient concentration in the uterus, and therefore probably originated from the female rather than from the male. This chemical stimulus for inducing mobility in the sperm may, for instance, be produced by the unfertilized oocytes. As mentioned in the introduction Anya (1973a, b) has postulated that 5 - hydroxytryptamine from the vas deferens of male A. tetraptera may initiate contractions of the female reproductive tract which help to bring the gametes together. Certainly, 5 - hydroxytryptamine has been shown to initiate vulval contraction in C. elegans and some other nematode species but also so has adrenaline (Croll, 1975). The present work has shown vigorous contractions of vulval/vaginal muscles in female P. redivivus after copulation but has also shown that the chains of sperm are capable of independent movement along the uterus. The role played by such contractions in fertilization in nematodes remains uncertain. In the female of most nematode species the sperm are stored 131 in the so-called seminal receptacle where they are generally attached to the walls by their pseudopodia (Lee, 1971; Anderson and Darling, 1964). In P. redivivus, however, the sperm were not stored in the "seminal receptacle", where fertilization generally took place, but remained in the uterus together with the developing embryos. Only the anterior-most sperm in the chain, those producing pseudopodia, entered the seminal receptacle for fertilization, and they remained part of the chain until just before fertilization and did not adhere to the walls of seminal receptacle. Even in the post-vulvar uterine sac, sometimes known as the posterior seminal receptacle (Hyman, 1951), the sperm never attached themselves to the walls. Chain formation of sperm in the uterus of female P. redivivus appeared not to be due solely to the suction pressure created by the pseudopodia, because the sperm remained in chains even when they were rounded with no pseudopodia visible. It seemed likely, therefore, that some other mechanism was involved, possibly secretions from the anterior and/or posterior regions of the sperm. Furthermore, sperm in the posterior sac did not form chains even when pseudopodia eventually appeared and this suggested some environmental influence on chain formation. Chain formation has only been reported in the sperm of one other nematode species, by Anderson and Darling (1962, 1964) in D. destructor, although many other species have been found where the sperm attach themselves singly to the walls of the seminal receptacle (Foor, 1970). The formation of chains of sperm in the uterus of P. redivivus was a very peculiar phenomenon characterised by all the sperm being connected anterio-posteriorly, the anterior end facing the seminal receptacle. 132 Observations on the sperm of P. redivivus have also shown that pseudopodial production decreased with age and ceased completely within 8 hours of insemination. The fact that pseudopodial production recommenced on reaching the seminal receptacle again suggested that pseudopodial production was influenced by secretions from the unfertilized oocytes. It seems reasonable to speculate that the unfertilized oocytes in P. redivivus females may be secreting some substance into the uterus forming a concentration gradient. This gradient being dependent upon the number of oocytes and their duration in the oviduct. Virgin females would have a strong concentration gradient due to the large number of oocytes present, whereas egg-producing females, with only a few oocytes for a relatively short time, would have a weaker concentration gradient. When the virgin female copulated all the sperm would produce pseudopodia because of the presence of unfertilized oocytes in the oviduct and they would move anteriorly along this concentration gradient. As the number of unfertilized oocytes decreased the sperm would produce fewer pseudopodia and this in fact appears to be the case. In normal, egg-producing females the concentration of the secretions would be lower and this could explain the observation of only 6 - 7 of the anterior-most sperm in the chain producing pseudopodia, as these would be the closest to the unfertilized oocytes in the seminal receptacle. However, this must remain speculative as in vitro experiments designed to examine the effect of unfertilized oocytes on pseudopodial production were inconclusive (Section VI.E.). 133 Copulatory behaviour of male nematodes It seems clear from the present experiments that the copulatory behaviour of male P. redivivus was largely influenced by its "physiological readiness" to copulate, or libido. Immediately after copulation males did not coil around another female; the frequency and duration of coiling increasing as the interval between two successive copulations increased. This increase in pre-copulatory coiling was probably due in part to the accumulation of large numbers of sperm in the seminal vesicle of the isolated male nematodes. However, it seems that copulation was also influenced by the presence or absence of the bubble-like structures in the posterior region of the vas deferens of P. redivivus. Male P. redivivus which had fully developed, large bubble-like structures copulated immediately, whereas those with less well developed structures took some time to copulate even if their seminal vesicle was over-loaded with sperm. In long-isolated males the bubble-like structures were variable in size and those males with small bubbles took longer to copulate than those with well developed bubbles (Section III.E.). In old males, incapable of copulation, the vas deferens was generally distorted and it is possible that these bubble-like structures cannot then develop in this region. Male P. redivivus normally copulated every 2 - 3 hours and released 20 - 30 sperm per copulation (Figs. 24 and 25). The results in Fig. 28.b. show that there was a rapid increase in the number of sperm released up to 12 hours of isolation, where it reached an 134 apparent maximum. This may be due.partly to the fact that the male has a limited space in which to store its sperm. The rather variable number of sperm released by males isolated for more than 12 hours may have been due to individual variations in the size of the seminal vesicle. Another factor limiting the number of sperm released at insemination may be the size of the female's uterus. Isolated males tended to release large amounts of fluid from the vas deferens into the uterus prior to the sperm and this may have prevented all of the seminal fluid and sperm present in the seminal vesicle of the male from entering the uterus. On the other hand, the male may not have been able to discharge the whole contents of its seminal vesicle. It is probable that an increased sensitivity to tactile/ chemosensory stimulation in males physiologically ready to copulate resulted in the observed indiscriminate coiling around females, other males, or even larvae (Section II.D.1.). When previously isolated males coiled around an attractive female they held the female very firmly with 2 - 3 coils of their body. Thus despite the increased copulatory activity of isolated males, the increased coiling reduced movement of the female through the coils and this had the effect of increasing the average vulval location time of the male. The increase in the duration of post-insemination coiling in isolated male P. redivivus (Fig. 28.b.) may have been due to their inability to regain their normal posture immediately after releasing a large amount of semen. This was reflected in the time taken for isolated males to withdraw their spicules from the vagina of the female, even if the female moved out of the coil. 135 The somewhat larger number of sperm released per copulation during the middle stages of the life span of a normal male P. redivivus (Fig. 25) may be due to the maturation of larger numbers of sperm in this period and/or to the observed increase in the size of the seminal vesicle with age, resulting in increased numbers of sperm which could be released at copulation. It is well established that nematode sperm mature both structurally and functionally in the uterus of the female (Section III.E.3.) and the isolated male experiments with P. redivivus clearly showed that the sperm did not lose their fertilizing capacity on storage in the seminal vesicle of the male (Fig. 28.c.). The sperm in the seminal vesicle appeared inactive and were presumably in a quiescent or reduced metabolic state with little energy utilization. Finally, although a direct comparison between virgin and normally copulating males P. redivivus was not made, the fact that of the five normally copulating males examined in the Section III.B.2. all survived for at least 9 - 10 days compared with 5 - 6 days for virgin males (Section III.C.2.) suggested that copulation tended to increase the life span of the male nematode. Sexual development of adult female nematodes Oocyte production in virgin female nematodes will obviously 'be influenced by the quality and quantity of the diet as well as by other environmental factors such as temperature. In non-virgin females the number of copulations also appears to influence oocyte 136 production in P. redivivus at least partly because this induces the female to feed more. The oocytes in the oviduct of female P. redivivus were shown to retain their ability for fertilization for a considerable time (Fig. 41). When the oocytes passed into the uterus this protective environment was lost and degeneration was found to be relatively rapid. As oocytes aged they eventually lost their capacity for fertilization and division, and aged oocytes in the uterus of female P. redivivus were very often unfertilized or if fertilized divided very slowly and failed to develop normally. All the unfertilized oocytes which passed into the uterus of P. redivivus remained there and degenerated. This suggests that ovoviviparous nematodes such as P. redivivus do not lay unfertilized eggs, unlike some other species, such as Pristionchus aerivora (Merrill and Ford, 1916). The storage of large numbers of oocytes in virgin female P. redivivus for a relatively long time before they started to degenerate (Fig. 31) as well as the storage of embryos and larvae for 20 - 24 hours after fertilization of oocytes in the copulated female (Fig. 32) suggests that there may be some chemical released by the developing larvae which forces the uterus to contract and expel the larvae out of the uterus. Another possibility, that the large number of eggs or larvae in the uterus forced the older eggs or larvae through the vulva appeared less likely in P. redivivus, as 48 hour old virgin females on copulation retained 31 ±7 larvae for the same length of time before laying as a newly moulted copulated female retained 20 1: 5 (Fig. 44). This suggested that the number 137 of eggs/larvae present was not the most important factor in controlling the birth of the larvae. Moreover,as the normally copulating female aged,it retained fewer larvae (Fig. 32) and died with only 6 - 7 larvae in its uterus. At insemination secretions from the vas deferens of P. redivivus generally passed into the post-vulvar uterine sac although sperm and seminal fluid usually passed into the uterus. It may be that this fluid from the vas deferens suppressed the production and/or collection of the fatty substance found only in the post-vulvar uterine sac of virgin females (Section IV). Since virgin females did not have sperm in their seminal receptacle to fertilize oocytes they had a very slow rate of oocyte production compared to normally copulating females, which presumably leads to the storage of excess food in the body wall and in the walls of the intestine. This food reserve may be used when the female stops feeding. Moreover, some of the oocytes from the oviduct were eventually reabsorbed, and this would provide another . source of energy which might help to increase their life span. Certainly, some virgin female P. redivivus survived for a much longer time than copulating females (Fig. 33) although this may . have been due to many other factors. Similar observations were also made by Fisher (1972) and Gaston, Shorey and Platzer (1974) on A. avenae and Rhabditis sp, respectively, where they also found that the life span of virgin females was much longer than that of normally copulating females. 138 Sex attraction in nematodes Adult females of P. redivivus were similar to other nematodes studied in secreting a sex attractant. However, fourth- stage larval females or females during the final moult neither attracted adult males nor responded to their attractant (Fig. 36). This behaviour is of course quite in accord with their development as fourth-stage larval females do not copulate. Cheng and Samoiloff (1971) on the other hand have claimed that fourth-stage larval females of P. silusiae attracted adult males, and Windrich (1973) reported that while fourth-stage larval female D. dipsaci did not attract adult males, adult males did attract fourth-stage larval females. However, it is likely that some of the fourth-stage larval females selected by Cheng and Samoiloff for the production of sex attractant would have moulted during the course of experiment (total duration 32 hours) and that the results they obtained might therefore have been due to these newly moulted adult females rather than the fourth-stage larval females. In the present work, preliminary experiments using randomly selected fourth-stage larval female P. redivivus also showed sex attraction after 32 hours incubation, by which time more than 50% of the larval females had moulted. When only early fourth-stage females were used no moulting occurred before the end of the experiment and no sex attraction was observed (Fig. 36). The experiments conducted by Windrich have similar shortcomings because according to the author the females reached the area of the males between 12 days and 4 weeks after inoculation, and 139 it is probable that some of the fourth-stage larval females would have moulted to the adult stage during this period. This could again explain the observed movement of the female towards the male rather than the reverse which is more common in nematodes (e.g. P. redivivus, Fig. 39.b.)., as the male would probably have secreted attractant during the whole experimental period, unlike the female. It has also been shown that virgin females of P. redivivus attract and respond to adult males, whereas copulated females neither attracted nor responded to the adult males (Fig. 36). When the fourth- stage larval female moults to the adult stage the ovary starts maturing oocytes, which are stored in the oviduct in the virgin female but which in the copulated female are fertilized in the seminal receptacle and are passed into the uterus. It seems probable that the presence of oocytes in the oviduct and the lack of sperm in the seminal receptacle make virgin female P. redivivus attractive to and respondent towards adult males, unlike copulated females. It is likely that sex attraction has evolved separately in different groups of nematodes in relation to their life cycle. For example, in the primitive form of sex attraction both sexes would probably secrete sex attractants. In more specialized nematodes only one sex might produce an attractant, such as in the genera Heterodera and Globodera, where the females are sedentary and need to be highly attractive to their males, while males do not need to be attractive. Similarly, in some groups of insects both sexes produce an attractant while in other groups only the male or the female does so (Jacobson, 1972). 140 It is important to note that female nematodes only copulate a few times, whereas male nematodes copulate frequently. In fact males of P. redivivus were observed to copulate 10 - 12 times per day and about 70 - 100 times in their lifetime (Fig. 24), whereas female P. redivivus only copulated 4 - 5 times in their lifetime, averaging about one copulation per day (Fig. 42). These results together with observations on A. tetraptera (C.L. Duggal, unpublished data) suggest that male nematodes are almost always ready to copulate showing a response towards the female sex attractant, whereas this is not the case with female nematodes. The latter only attract and respond when they have no sperm and large numbers of ooctyes stored in the oviduct. Moreover, in species which lay large numbers of eggs (or larvae) during their lifetime, as is the case with most animal parasitic nematodes, it is likely that the oviduct contains oocytes irrespective of whether they are copulated or not. In these species it might be expected that the females are attractive to adult males for their entire adult life, and this has in fact been observed in Ancylostoma caninum (Roche, 1966). Sex attraction experiments on animal parasitic nematodes have invariably been conducted with non-virgin females and the failure to detect a female response towards males in these species may have been due to the females containing sperm from a previous copulation, and it might be expected that if virgin females were examined attraction would be found. The present studies on the development of the female reproductive system in P. redivivus in relation to sex attraction 141 have shown that complete development of the gonads was not sufficient for the production of sex attractant as it also appeared to depend upon the number of unfertilized oocytes stored in the oviduct (Figs. 37 and 38). Virgin female P. redivivus,which did not have sperm in the seminal receptacle, stored large numbers of oocytes in the oviduct and were found to produce a sex attractant, whereas copulated females, which had very few oocytes in the oviduct, produced little or no sex attractant. These experiments clearly suggest that the production of sex attraction by female P. redivivus does not depend upon the regular production of oocytes but depends upon the storage of oocytes in the oviduct. It may be supposed that the sex attractant or its precursor is produced by the unfertilized oocytes in the oviduct and that the sex attractant passes into the external environment through the general body surface of the nematode, principally via the vulval aperture. Little is known about the receptor site(s) for sex attraction in nematodes. Aldicarb,a carbamate pesticide, has been shown to disrupt sex attraction in H. schachtii at very low concentrations (Hough and Thomson, 1975) and this suggests that it is acting on a sense organ(s) which uses acetylcholine as a neurotransmitter by inhibiting the enzyme acetylcholinesterase (D.J. Wright, pers. comm.). Certainly, acetylcholinesterase activity has been located in the amphids, phasmids and cephalic papillae of D. viteae (McLaren, 1972) and C. elegans (Pertel, Paran and Mattern, 1976) and in other regions of the nematode nervous system (Wright and Awan, 1976). The presence of esterase activity in the spicules of some 142 nematodes indicates that they could also be involved in sex attraction. Copulatory behaviour of female nematodes The results in Section VI suggest that no post-moulting maturation period was required before insemination of female P. redivivus could occur,, at least at 25°C, the timing of the first ircemination being determined largely by the number of males present in the immediate vicinity of the moulting female (Fig. 40). As mentioned previously virgin female P. redivivus remained attractive to the adult males for most of their life, possibly due to the presence of large numbers of oocytes in the oviduct. When the females stopped feeding no further copulations occurred and the female eventually died. When a female P. redivivus copulated for the first time with a normal male (taken from a culture where it copulated ad libidum) the female received 20 - 30 sperm which rapidly reached the anterior part of the uterus and fertilized the oocytes in the seminal receptacle. Normal females kept at 25°C produced 24 - 30 eggs in the 24 hours after copulation and each egg was fertilized by one sperm. If females only copulated again after all the sperm had been consumed then the second copulation should occur 20 - 30 hours after the first copulation. However, this was not the case and some females copulated with sperm remaining in their uterus. This was particularly common where the female received more than 80 sperm from a previously 143 isolated male (Fig. 43). This may have been due to the rapid aging of sperm once they were released into the uterus, where as mentioned previously they become structurally and functionally mature. After 20 hours from the previous copulation an increasingly large percentage of females copulated with sperm still in their uterus. It is also possible, however, that some substance was released by functionally mature sperm into the uterus which was involved in the further delaying ofr copulation, although there is as yet no evidence for this. The aging of sperm in the uterus of the female was the major factor leading to the second copulation in female P. redivivus receiving more than 80 sperm at the first copulation (Fig. 43). However, this was not the case with females which received normal numbers of sperm. The fact that a high percentage of females copulated only after consuming the sperm from the first copulation suggested that these sperm did not rapidly lose their capability for fertilization, at least not within 24 hours of the first copulation (Fig. 43). At the third copulation in P. redivivus, where the female was not much older than at the second copulation and which probably produced.00cytes at the same rate, a similar situation applied with most females only copulating again after consuming the sperm from the previous copulation (Fig. 47). Another copulation may occur'when too few sperm are present in the female, or when sperm lose their ability to fertilize oocytes leading to an accumulation of oocytes in the oviduct, which may as mentioned earlier secrete substances favouring copulation in 144 P. redivivus. There is also the possibility that substances released at the time of copulation may be playing a part in preventing further copulations, the amount released depending upon the number of sperm transferred. Long isolated males which transferred more than 80 sperm, released larger amounts of fluid from the vas deferens than normally copulating males. On the other hand some females copulated again within a few minutes of their first copulation and this may have been due to the female taking some time to react to the initial copulation. Two successive copulations were very common in female P. redivivus which had large numbers of oocytes in their oviduct and were not infrequent in females with no more than a normal number of oocytes. However, this was only found when the females received normal numbers of sperm, when large numbers of sperm were given further copulations did not occur for at least 12 hours (Fig. 42): This may have been due to the small number of sperm (and/or the small amount of semen) received being insufficient to inhibit further copulations. The results in Figure 46 show that the number of oocytes also affected the rate of copulation. As already mentioned, newly moulted females produced about 24 - 30 oocytes in 24 hours at 25°C, enough to consume the sperm received at a "normal" insemination; but if a female already had oocytes in the oviduct there was a more rapid consumption of sperm (Figs. 44 and 45) and in this case the female tended to copulate again more rapidly (Fig. 46). In 48 hour old virgin femalds, for example, 30 - 40 oocytes were fertilized within 16 hours of copulation (Fig. 44). The rates of fertilization of oocytes 145 in females receiving high or low numbers of sperm were similar (Fig. 44) indicating that the number of oocytes was the limiting factor in fertilization.. Aging of oocytes was another important factor, oocytes appeared to age very quickly in the uterus of female P. redivivus, starting to degenerate within a few days if they were not fertilized by sperm. These agdd oocytes probably did not affect the rate of copulation significantly. The oocytes in the oviduct, however, remained viable for a long time, aging only slowly. One or two day old virgin females were able to copulate quickly but as they aged their oocytes may not have been able to consume all of the sperm present leading to longer intervals between two successive copulations (Fig. 46). Aging in female nematodes generally may play an important role in determining the interval between copulations. As for example in the case of an ovoviviparous species, such as R. pellio where the rate of larval production declines with age (Somers, Shorey and Gaston, 1977). These females may not consume as many sperm as the young copulating females and this again may lead to an increase in the time between copulations. However, if secretions from the oocytes play a part in initiating copulatory activity, the decline in oocyte production with age would also lead to a decline in the rate of copulation. Physical conditions may also play some role in copulation in nematodes, for exampler in aging females the muscles of the vagina and the vulva may not allow the spicules of the male to penetrate readily and so prevent or delay insemination. 146 CONCLUSIONS P. redivivus is a free-living saprophagous nematode capable of very rapid reproduction given favourable environmental conditions, with a generation time of 3 - 4 days at 25°C. It is thus an r-selected type of organism (Pianka, 1970) tending to have a widely fluctuating population, rapidly colonizing new and often short-lived habitats. Female P. redivivus were found to lay between 80 - 90 larvae during their life span and copulated about 4 - 5 times. Egg production was greatly influenced by the quantity and quality of food as females started to mature more oocytes when food, was plentiful, and the presence of unfertilized oocytes in the oviduct made the 'female attractive to and responsive towards males. As both the male and female in this species are motile it seems unlikely that sex attraction would be particularly important in bringing the two sexes together, except perhaps when they are in close proximity to each other. However, as the female was also responsive towards males any random movement which brings the two sexes together would lead to copulation and also to aggregation of individuals of the same species. This aggregation phenomenon of sex attraction would increase the density of local populations which would further help in population growth. Moreover, under unfavourable conditions aggregated nematode populations may have a better chance of survival than isolated individuals. Aggregations may also result in better dispersal with the help of insects or other animals leading to the colonization of 147 more favourable habitats. Repeated copulations were found to be necessary to induce female P. redivivus to feed more and it was also important to supply the female with sufficient viable sperm. The latter appears to be an important limiting factor in this species as the number of sperm received at one copulation was only sufficient to fertilize the oocytes produced over a 24 - 48 hour period; excess sperm losing their fertilizing capacity and degenerating within this period. Phillipson (1969); Somers, Shorey and Gaston (1977) and Johnson, Orihel and Beaver (1974) have also concluded that females of other nematode species must copulate once every two days in order to maintain output of fertile eggs. The presence of unfertilized oocytes in the oviduct not only made female P. redivivus attractive to and responsive towards males it also facilitated insemination and rapid maturation of sperm inside the uterus, whereas in females with few oocytes, and sperm already in the uterus, insemination did not usually take place. This would tend to reduce wastage of available sperm. Moreover, male P. redivivus unable to copulate within 2 - 3 hours of the previous copulation continued to store sperm in the seminal vesicle where they remained viable and after insemination fertilized more oocytes, over a longer period than the sperm of a normal copulation. Obviously,the above factors will all aid in the exploitation of favourable conditions by rapid population growth. 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YUKSEL, H.S. (1960). Observations on the life-cycle of Ditylenchus dipsaci in onion seedlings. Nematologica 5, 289 - 296. 158 Appendix III.B. Fig. 24. .Rate of copulation per day during the adult life of male P. redivivus Replicate Days after final moult 1 2 3 4 5. 6 7 8 9 10 1 8 8 7 4 7 9 9 10 10 6 2 10 13 9 10 9 13 10 9 10 8 3 10 11 9 9 11 11 10 7 2 1 4 8 5 11 9 10 9 10 8 1 - 5 11 15 12 11 9 12 9 8 3 - Total 47 52 48 42 46 54 48 42 26 15 Mean 9.4 10.4 9.6 8.4 9.2 10.8 9.6 8.4 5.2 5.0 S.D. 1.3 4.0 1.9 2.5 1.5 1.8 0.5 1.1 4.4 3.6 159 Appendix III.B. Fig. 24. Rate of sperm transferred per day during the adult life of male P. redivivus Days after final moult Replicate 1 2 3 4 5 6 7 8 9 10 1 236 248 153 106 150 263 265 266 230 176 2 173 178 140 170 175 378 257 190 183 177 3 213 353 356 297 310 378 374 230 69 21 4 186 176 399 313 339 297 330 210 13 5 210 386 390 326 356 515 421 234 84 Total 1018 1341 1438 1212 1330 1831 1647 1130 579 374 Mean 203.6 268.2 287.6 222.4 266.0 366.2 329.4 266.0 115.8 124.7 S.D. 24.6 97.6 129.9 98.5 96.3 97.3 70.3 28.4 88.5 89.8 160 Appendix Fig. 27. Mortality rate of virgin male P. redivivus Days after final moult Replicate 1 2 3 4 5 6 1 12 23 35 43 46 49 2 12 26 38 47 49 50 3 11 16 26 41 48 50 4 13 17 25 39 46 49 Total dead 48 82 124 170 189 198 % mortality 24 41 62 85 94.5 99 S.D. 1.6 9.6 13.0 6.8 3.0 1.2 161 Appendix III.D.2a. Fig. 28.a. The effect of short separation from females on the duration (minutes) of pre-insemination coiling of male P. redivivus Isolation period (hours) after final moult Replicate 6 12 18 24 30 1 2 4 7 8 2 2 4 6 10 2 1 3 3 5 2 5 17 4 8 2 6 5 3 5 - 11 3 15 12 6 - - 2 Total 17 28 30 35 35 Mean 4.25 5.6 5.0 7.0 7.0 S.D. 2.6 3.4 3.2 4.9 7.2 Regression line was drawn and significance of slope was compared y = a 4- a x ao = 4.45 o 1 = 0.08 = 4.45 + 0.08x a1 r2 = 0.02 t = 0.08 0.12 Sy.x = 4.37 = 0.666 S = 2.30 o S = 0.12 P = > 0.1 1 n = 25 df = 23 162 Appendix III.D.2b. Fig. 28.b. The effect of short separation from females on the number Of sperm transferred at copulation of male P. redivivus Isolation period (hours) after final moult Replicate 6 12 18 24 30 1 68 113 55 86 63 2 63 82 67 71 97 3 69 .112 84 102 82 4 73 105 91 76 154 5 - - 76 65 129 6 - - 122 - - Total 273 412 495 400 525 Mean 68.25 103.0 82.5 80.0 105.0 S.D. 4.1 14.4 23.1 14.5 36.5. - 163 Appendix III.D.2c. Fig. 28.b. The effect of short separation from females on the duration (seconds) of post-insemination coiling of male P.'redivivus Isolation period (hours) after final moult Replicite 6 12 18 24 30 1 60 112 315 360 285 2 69 249 210 240 267 3 80 184 235 360 292 4 - 156 167 240 165 5 - 190 - Total 209 891 927 1200 1009 Mean 69.7 178.2 231.7 300.0 252.2 S.D. 10.0 50.1 62.1 69.3 59.1 164 Appendix III.E. Fig. 28.c. The effect of long isolation periods of virgin male P. redivivus on the % of males copulating in' an hour period. Isolation period (hours) after final moult Replicate 24 48 72 96 1 7 8 3 4 2 7 5 4 3 3 8 7 4 3 Total 22 20 11 10 % copulated 73 67 37 33 Regression line was drawn and significance of slope was compared. a 90.0 y = a a x o = o 1 = a1 -0.63 = 90 - 0.63x 2 r = 0.76 t = -0.63 0.11 Sy.x = 10.25 = 5.727 S = 0 7.25 S1 = 0.11 P = < 0.001 n = 12 df = 10 165 Appendix III .E Fig. 28.c. The effect of prolonged storage of sperm in the seminal vesicle of the male on the number of oocytes fertilized by them in the following 24 hours of insemination Age of sperm (hours) and oocyte fertilized RepliCate 24 48 72 96 1 •30 21 19 19 2 15 . 13 11 9 3 12 21 21 15 4 26 11 21 33 5 17 31 13 14 6 16 21 18 29 7 15 11 23 21 8 20 28 15 17 9 18 26 14 23 10 i 14 20 - 18 11 28 14 - 12 7 23 - 13 13 10 - 14 23 35 15 25 14 - 16 15 23 - 17 23 14 - 18 .19. - - 19 27 - - 20 19 - 21 29 - - 22 24 - - Total 435 336 155 198 Mean 19.77 19.76 17.22 19.8 S.D. 6.22 7.44 4.15 7.11 Regression line was drawn and significance of slope was compared a = o 19.97 y = ao 4- a1x a = -0.01 1 = 19.97 - 0.01x 2 r = 0.00 t = -0.01 Sy.x = 6.46 0.03 S o = 1.84 0.33 S = 0.03 1 P = > 0.1 n = 58 df = 56 •166 Appendix IV.A.3a. Fig. 31 Storage of oocytes in the oviduct of the virgin female P. redivivus with age Days after final moult Replicate 2 3 4 5 6 7 8 9 10 1 4 24 35 40 42 46 28 21 12 10 6 2 .4 22 32 32 34 41 41 37 25 10 3 4 23 26 29 34 36 37 25 14 7 4 4 22 20 26 28 32 29 23 20 10 5. 4 24 28 30 40 41 30 23. 15 5 6 4 22 32 33 34 35 25 7 - - 7 4 23 25 27 28 30 24 15 12 7 8 4 19 28 30 39 30 30 26 - - - 9 3 18 30 35 44 40 35 - - - - 10 4 25 25 33 35 38 47 - - - 11 3 18 30 32 23 ------12 4 24 24 27 29 ------13 3 25 35 37 39 30 - - - - 14' 4 18 22 25 ------15 4 25 25 18 ------16 - 4 25 25 30 32 23 - - - Total 61 357 442 484 481 422 326 177 98 49 12 Mean 3.8 22.3 27.6 30.2 34.4 35.2 32.6 22.1 16.3 8.1 6.0 S.D. 0.4 2.65 4.4 5.2 6.0 6.44 7.3 8.68 5.16 2.14 0 167 Appendix IV.A.3b. Fig. 31. Storage of oocytes in the oviduct of normally copulating female P. redivivus with age Days after final moult Replicate 0 1 2 3 4 5 6 1 4 20 15 0 - 2 4 20 17 5 4 3 3 3 13 17 4 2 2 4 3 3 - - - 5 4 22 0 - - - 6 4 18 17 8 3 3 7 4 19 13 7 3 2 8 4 19 26 - - - 9 4 21 0 - - 10 3 17 15 5 - Total 37 172 120 34 14 10 2 Mean 3.7 17.2 13.3 5.7 3.5 2.5 S.D. 0.5 5.57 8.38 3.2 0.6 0.58 168 Appendix IV.A.3a. Fig. 32. Storage of oocytes in the uterus of the virgin female P. redivivus with age Days after final moult Replicate 0 1 2 3 4 5 6 7 8 9 10 1 '0 3 12 18 31 48 62 59 55 34 28 2 0 2 12 15 20 28 29 36 24 0 - 3 0 4 12 19 28 37 26 20 0 0 - 4 0 4 19 23 25 30 35 38 20 14 10 5 0 2 8 14 19 25 20 18 16 13 - 6 0 5 10 14 26 35 37 23 - - - 7 0 4 13 14 25 33 23 20 13 7 - 8 0 2 8 14 28 27 20 9 - - - 9 0 3 8 15 28 33 31 - - - - 10 0 2 8 20 35 33 36 - - - - 11 0 2 10 17 29 ------12 0 4 20 21 37 ------13 0 2 15 15 24 29 - - - - - 14 0 4 5 12 ------15 0 4 22 33 ------16 0 4 5 15 25 38 - - - - - Total 0 51 187 279 380 396 319 223 128 68 38 Mean 0 3.19 11.69 17.44 27.14 33.0 31.9 27.9 25.6 17.0 19 S.D. - 1.05 5.11 5.14 4.97 6.18 12.3 15.76 16.95 11.75 169 Appendix IV.A.3b. Fig. 32. Storage of embryos and larvae in the uterus of normally copulating female P. redivivus with age Days after final moult , Replicate 0 1 2 3 4 5 6 r 0 44 47 7 7 2 0 45 42 8 7 8 3 0 33 30 26 13 4 11 4 0 29 12 - - - - 5 0 45 21 8 - - - 6 0 42 35 21 15 8 7 0 44 45 15 29 7 8 0 54 40 13 - - - 9 0 38 18 - - - 10 0 29 15 18 7 - - Total 0 403 305 116 78 27 11 Mean 0 40.3 30.5 14.5 13.0 6.75 S.D. - 8.0 13.14 6.87 8.58 1.9 - Appendix IV.A.3c. Fig. 33. Mortality rate of virgin female P. redivivus Days after final moult Replicate 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1 3 9 16 30 41 54 66 75 80 88 90 91 93 95 95 98 99 100 100 100 2 3 13 17 26 38 51 60 68 75 82 88 92 95 96 96 98 98 98 100 100 3 2 8 18 24 35 45 55 63 74 84 89 92 92 93 96 97 98 98 98 99 4 1 7 10 12 32 49 58 71 85 90 94 98 98 99 100 100 100 100 100 100 5 0 4 13 26 34 38 40 48 64 70 78 84 88 90 90 92 92 94 95 97 6 2 7 11 14 30 38 44 59 69 85 90 93 94 95 96 97. 98 99 99 99 7 0 3 5 9 24 32 37 49 65 80 84 87 88 91 93 95 97 97 97 98 8 3 8 13 20 32 45 54 59 72 83 89 94 94 95 96 96 97 98 99 99 9 1 7 12 20 33 44 51 61 73 82 87 91 93 94 95 97 98 98 99 100 10 2 7 13 20 33 43 51 60 72 82 87 90 92 93 94 97 97 98 99 99 Total dead 17 73 128 201 332 439 516 613 729 826 876 912 927 941 951 967 974 980 986 991 % mortality 1.7 7.3 12.8 20.1 33.2 43.9 51.6 61.3 72.9 82.6 87.6 91.2 92.7 94.1 95.1 96.7 97.4 98.0 98.6 99.1 S.D. 1.2 2.3 3.8 6.7 4.5 6.6 9.1 8.6 6.3 5.4 4.2 3.8 3.0 2.6 2.6 2.1 2.1 1.7 1.6 1.0 171 Appendix IV.A.3c. Fig. 33. Mortality rate of normally copulating female P.'redivivus Days after final moult Replicate 2 3 4 5 6 7 8 9 10 11 1 0 5 18 30 48 64 83 92 98 100 100 2 0 9 21 29 49 62 81 93 98 100 100 3 0 4 13 20 22 39 66 82 96 100 100 4 1 6 13 20 21 36 54 81 93 98 100 5 0 3 8 10 20 39 68 86 97 100 100 Total dead 1 27 73 109 160 240 352 434 482 498 500 % mortality 0.2 5.4 14.6 21.8 32.0 48.0 70.4 86.8 96.4 99.6 100 S.D. - 2.3 5.0 8.1 15.1 13.8 11.9 5.5 2.1 0.9 0.0 172 Appendix V.C.1. Fig. 35.b. Distribution of male P. redivivus on agar strips-after- -6 hours -at-25°C in-response-to- E coli Number of worms per zone Replicate (E)x3 x2 xl . 0 yl y2 y3(E) 1 31 8 6 29 9 2 15 2 - 21 1 4 61 1 4 8 3 31 4 3 44 7 2 9 4 17 4 3 42 3 5 26 5 13 3 5 51 6 5 17 6 9 2 3 57 1 3 25 7 21 3 5 33 8 2 28 8 18 3 9 31 5 4 30 9 24 16 7 27 5 4 17 10 17 5 4 32 4 8 30 Total 202 49 49 407 49 39 205 Student's t-test Chi-square test 2 s = 97.45 X2 = 24.5 t = 0.16 P =, > 0.05 P = > 0.1 173 Appendix V.C.1. Fig. 35.d. Distribution of newly moulted virgin female P. redivivus on agar strips after -6 hours at 25°C in response to E. coli. Number of worms per zone Replicate (E)x3 x2 xl 0 yl y2 y3(E) 1 16 7 4 35 10 1 27 2 6 3 5 54 8 6 18 3 10 3 7 20 8 5 47 4 15 5 1 53 4 6 16 5 35 5 3 29 2 5 21 6 24 10 4 49 3 3 17 7 17 6 3 44 3 5 22 8 32 4 1 40 4 3 16 9 26 6 5 43 3 . 6 11 10 29 2 3 36 5 5 10 Total ' 210 51 36 403 50 45 205 Student's t-test Chi-square test 2 2 s = 116.339 = 21.86 t = 1.315 P = > 0.05 P = > 0.1 174 Appendix V.C.2. Fig. 35.a.' Distribution of male P. redivivus on agar strips after 6 hours at 25°C in response to males Number of worms per zone Replicate (E)x3 x2 xl 0 yl y2 y3(E.1006) 1 21 4 3 44 3 5 20 2 33 6 2 23 2 3 31 3 19 4 5 38 5 2. 27 4 16 6 4 43 4 7 20 5 17 6 5 41 4 4 23 6 20 1 4 29 5 4 37 7 24 4 3 31 4 6 28 8 29 5 2 40 2 3 19 9 23 4 3 32 5 3 30 10 22 5 7 33 4 5 24 Total 224 45 38 354 38 42 259 Student's t-test Chi-square test 2 2 s = 30.944 ')4.. = 3.458 t = 1.286 > 0.995 P = > 0.1 175 Appendix V.C.2. Fig. 35.c. Distribution of virgin female P. redivivus on agar strips after 6 hours at 25°C in response to virgin females. Number of worms per zone Replicate (E)x3 x2 xl 0 yl y2 y3(E.100 ) 1 32 4 2 36 1 2 23 2 1,9 4 2 52 1 4 18 3 21 5 2 32 5 1 34 4 23 6 5 38 3 3 22 5 28 1 1 40 5 4 21 6 27 4 2 37 1 2 27 7 33 2 5 36 2 3 19 8 22 3 3 31 3 4 34 9 27 3 5 31 3 5 • 26 10 25 0 2 36 2 6 29 Total 257 32 29 369 26 34 253 Student's t-test Chi-square test 2 s = 33.206 7C2 = 5.763 t = 0.194 P = > 0.995 P = > 0.1 176 Appendix V.C.3. Fig. 36.a. Distribution of male P. redivivus on agar strips after 6 hours at 25°C in response to fourth stage females Number of worms per zone Replicate (E)x3 x2 xt 0 yl y2 y3(E.1009) 1 20 2 4 38 3 3 30 2 33 2 4 24 3 4 30 3 24 8 5 31 5 3 24 4 17 1 8 37 10, 3 24 5 22 6 9 27 4 5 27 6 28 4 4 28 5 0 31 7 22 10 7 29 7 3 22 8 16 2 9 38 6 0 29 9 23 3 6 37 6 1 24 10 23 2 7 47 1 0 20 Total 228 40 63 336 50 22 261 Student's t-test Chi-square test 2 s 25.611 ..;42 0.038 0.017 P = > 0.995 P = > 0.1 177 Appendix V.C.3. Fig. 36.b. Distribution of fourth stage femdle P. redivivus on agar strips after 6 hours at 25°C in responSe to males Number of worms per. zone Replicate (E)x3 x2 xl 0 yl y2 y3 (E.100d ) 1 '38 4 2 26 2 6 22 2 51 3 3 15 0 4 24 3 25 6 1 22 0 1 45 4 13 4 4 35 3 8 33 5 30 3 2 31 1 1 32 6 29 2 2 22 3 1 41 7 ' 22 3 3 38 1 6 27 8 18 7 6 28 6 4 31 9 15 3 9 44 3 6 20 10 19 3 11 27 6 5 29 Total 260 38 43 288 25 42 304_ Student's t-test Chi-square test 2 s = 73.878 "k2 . 10.6 _ t 0.780 > 0.05 P = > 0.1 178 Appendix V.C.3. Distribution of male P. redivivus on agar strips after 6 hours at 25°C in response to mixed stages of larval fourth stage, moulting and moulted adult females. Number of worms per zone Replicate (E)X3 x2 xl 0 yl y2 y3(E.100?) 1 15 1 1 29 1 0 53 2 13 5 4 37 3 6 32 3 10 7 14 8 3 56 4 18 2 3 9 1 8 59 Total 56 10 15 89 13 17 200 Student's t-test Chi-square test 2 s 84.625 2 = 105.39 t = 5.73 < 0.001 P = < 0.002 179 Appendix V.C.4. Fig. 36.c. Distribution of male P. redivivus on agar strips after 6 hours at 25°C in response to newly copulated females Number of worms per zone Replicate (E)x3 x2 xl 0 yl y2 y3(E.100?) 1 13 4 6 35 3 3 36 2 8 3 7 39 2 3 38 3 16 7 6 48 1 3 19 4 10 1 2 69 3 1 14 5 27 3 5 38 3 1 :23 6 17 3 7 41 4 2 26 7 26 6 4 22 2 9 31 8 14 2 8 23 5 5 •43 9 13 12 6 34 2 5 28 10 19 10 6 28 5' 8 24 Total 163 51 57 377 30 40 282 Student's t-test Chi-square test 2 s 84.806 )C? = 18.424 t = 1.97 P = > 0.05 P = > 0.05 180 Appendix V.C.4. Fig. 36.d. Distribution of newly copulated female P. redivivus on agar strips after 6-hours at 25°C- in response to males Number of worms per zone Replicate (E)x3 x2 xl 0 yl y2 y3(E.100d) 1 22 9 6 29 3 5 26 2 34 11 11 21 5 1 17 3 20 7 5 33 2 2 31 4 21 2 7 34 5 5 26 5 25 1 4 38 4 3 25 6 18 4 5 36 4 6 27 7 24 6 8 38 5 1 18 14 6 11 43 6 3 17 9 18 7 7 33 4 5 26 10 22. 5 8 21 3 12 29 Total 218 58 72 326 41 43 242 Students t-test Chi-square test 2 2 s = 54.778 .x.. = 0.968 t = 0.665 P > 0.995 P = > 0.1 181 Appendix V.C.4. Fig. 36.e. Distribution of male P. redivivus on agar strips after 6-hoUrs at 25°C - in response- to-gravid -females Number of worms per zone Replicate (E) x3 x2 xl 0 yl y2 y3 (E .100 9 ) 1 29 4 10 24 2 4 27 2 31 3 1 34 3 3 25 3 23 0 7 21 2 5 42 4 41 1 2 21 2 4 29 5 42 4 7 16 3 3 25 6 28 2 3 18 7 5 37 7 16 0 0 42 4 2 36 8 13 2 2 56 5 4 18 9 11 0 2 56 2 3 26 10 24 3 4 27 3 8 31 Total 258 19 38 315 33 41 296 Student's t-test Chi-square test 2 s = 118.139 = 6.05 = 1.315 P > 0.995 P = > 0.1 182 Appendix V.C.4. Fig. 36.f. Distribution of gravid female P. redivivus on agar strips after 6 hours at 25°C in response to males Number of worms per zone Replicate (E)x3 x2 xl 0 yl y2 y3(E.100 a ) 1 25 3 11 42 2 3 14 2 31 7 5 30 5 0 22 3 21 3 3 42 3 5 23 4 28 8 10 27 8 3 16 5 20 1 5 22 22 5 25 6 21 10 13 27 5 10 14 7 15 5 4 23 6 4 43 8 25 6 2 37 5 2 23 9 20 4 5 42 2 5 22 10 15 6 7 43 4 2 23 Total 221 53 65 335 62 39 225 Student's t-test Chi-square test 2 2 s = 95.183 0.266 t = 0.298 P = > 0.995 P = > 0.1 183 Appendix V.C.5. . Fig. 37.a. Distribution of male P. redivivus on agar strips after 6 hours at-25°Z in response to newly.moulted females . Number of worms per zone Replicate (E)x3 x2 xl 0 yl y2 y3(E.looy 16 4 3 34 3 7 33 2 10 1 2 30 4 0 53 3 12 1 1 43 4 .4 35 4 19 1 31 3 3 43 5 13 1 47 1 2 36 6 19 1 3 36 2 1 38 7 20 3 1 24 1 2 49 8 11 1 2 30 3 3 50 9 6 2 0 18 7 3 64 10 12 6 9 4 9 52 Total 138 21 20 302 32 34 453 Student's t-test Chi-square test 2 2 s = 82.322 ;4.. = 177.836 t = 8.379 P = < 0.001 P = < 0.001 184 Appendix V.C.5. Fig. 37.b. Distribution of male P. redivivus .on agar strips after 6 hours at 25°C in response to 48 hours old virgin females Number of worms per zone Replicate (E)x3 x2 xl 0 yl y2 y3 (E .100 Q ) 1 4 1 3 36 5 2 49 2 7 5 3 33 2 6 44 3 9 1 9 16 0 7 58 4 5 2 0 50 4 1 38 5 .11 3 5 43 1 3 34 6 14 3 2 45 1 0 35 7 6 2 0 54 2 1 35 8 7 1 0 42 9 6 35 9 7 3 4 26 6 6 48. 10 8 1 5 37 5 15 29 Total 78 22 31 382 35 47 405 Student's t-test Chi-square test 2 s = 61.5 -X = 200.78 t = 10.151 P = < 0.001 < 0.001 185 Appendix V.C.5. Fig. 37.c. Distribution of male P. redivivus on agar strips after 6 hours at 25°C in response to 96 hours old virgin females Number of worms per zone Replicate (E)x3 x2 xl 0 yl y2 y3 (E .100T ) 1 4 1 0 73 2 6 14 2 9 5 3 57 0 2 24 3 10 4 9 29 .4 4 40 4 11 3 5 52 1 2 26 5 13 3 3 27 15 5 34 6 14 0 2 46 2 6 30 7 6 5 6 45 4 4 30 8 15 2 7 17 6 10 43 9 9 3 1 41 9 3 34 10 19 6 6 29 8 5 27 . Total 110 32 42 416 51 47 302 Student's t-test Chi-square test 2 s2 = 96.467 7.. = 100.75 t = 4.92 < 0.001 P = < 0.001 186 Appendix V.C.5. Fig. 37.d. Distribution of male P. redivivus on agar strips after 6 hours at 25°C in response to 144 hours old virgin females Number of worms per zone Replicate (E)x3 x2 xl 0 yl y2 y3(E.100 ) 1 7 0 2 38 1 8 44 2 24 3 0 14 1 6 52 3 31. 3 5 27 4 5 25 4 26 4 6 21 2 6 35 5 41 3 2 19 2 1 32 6 11 4 2 59 1 2 21 7 3 4 1 48 2 9 33 8 19 4 1 19 7 2 48 9 35 1 3 10 3 6 42 10 37 5 2 7 4 3 42 Total 234 31 24 262 27 48 374 Student's t-test Chi-square test 2 s = 161.1 2 = 61.304 t = 2.82 < 0.001 P = < 0.02 187 Appendix V.C.5. Fig. 37.e. Distribution of male P. redivivus on agar strips after 6 hours at 25°C in response to 192 hours old virgin females NUMber of worms per zone Replicate (E)x3 x2 xl 0 yl y2 y3 (E do° 1 30 3 1 24 0 4 38 2 20 7 6 37 7 5 18 3 18 5 6 35 11 6 19 4 17 3 2 57 1 4 16 5 25 3 1 43 3 5 20 6 35 2 6 21 4 2 30 7 40 9 2 16 5 3 25 8 14 7 7 29 11 7 25 9 30 4 3 32 2 2 27 10 26 6 0 37 3 5 23 Total 255 49 34 331 47 43 241 Student's t-test Chi-square test 2 s = 55.444 = 0.098 t = 0.21 P = > 0.995 > 0.1 188 Appendix V.C.5. Fig. 38..a. Distribution of newly moulted virgin female P. redivivus on agar strips after 6 hours at 25°C in response to males Number of worms per zone Replicate (E)x3 x2 xl 0 yl y2 y3(E.100d) 1 8 1 2 45 2 4 38 2 12 2 4 29 3 6 44 3 19 8 9 20 9 7 28 4 12 5 3 22 2 7 49 5 21 4 3 31 2 3 36 6 8 4 5 14 4 7 58 7 18 7 3 .28 7 6 31 8 8 1 2 26 4 6 53 9 9 1 3 33 8 4 42 10 15 1 9 21 3 7 44 Total 130 34 43 269 44 57 423 .Student's t-test Chi-square test 2 2 s = 77.472 = 169.375 t = 8.053 P = < 0.001 < 0.001 189 Appendix V.C.5. Fig. 38.b. Distribution of 48 hours old virgin female P. redivivus on agar strips after 6 hours at 25°C in response to males Number of worms per zone Replicate (E)x3 x2 xl 0 yl y2 y3 (E .100 c3 ) 1 16 1 2 17 0 2 62 2 17 1 3 31 3 1 44 3 11 1 2 17 0 1 68 4 11 2 1 33 1 0 52 5 9 1 1 25 1 2 61 6 4 0 3 48 4 3 38 7 7 0 2 43 1 0 47 8 14 1 3 34 0 3 45 9 16 0 2 37 0 3 42 10 7 1 6 40 0 0 46 Total 112 8 25 325 10 15 505 Student's t-test Chi-square test 2 2 s 52.028 = 222.272 t = 11.935 < 0.001 P = < 0.001 190 Appendix V.C.5. Fig. 38.c. Distribution of 96 hours old virgin female P. redivivus on agar strips after 6 hours at 25°C in response to males Number. of worms per zone Replicate (E)x3 x2 xl 0 yl y2 y3 (E .100 d ) 1 29 3 2 10 1 0 55 2 11 1 0 26 1 0 61 3 36 1 0 10 1 2 50 4 17 1 0 4 2 0 76 .5 39 1 1 9 0 2 48 6 14 0 3 14 6 1 62 7 10 1 2 17 2 4 64 8 8 1 1 22 3 6 59 9 30 3 20 0 2 45 10 32 2 4 13 1 4 44 Total 226 11 16 145 17 21 564 Student's t-test Chi-square test 2 2 s = 97.317 "X, = 342.348 t = 7.911 < 0.001 P < 0.001 191 Appendix V.C.5. Fig. 38.d. Distribution of 144 hours Old virgin female P. redivivus on agar strips after 6 hours at 25°C in response to males Number of worms per zone Replicate (E)x3 x2 xl 0 yl y2 y3 (E .100 d ) 1 24 1 3 21 2 2 47 2 29 1 0 22 1 4 43 3 33 6 2 14 3 2 40 4 15 0 2 27 2 2 52 5 15 1 1 25 5 2 51 6 30 0 3 20 0 2 45 7 25 2 3 17 0 4 49 8 32 2 4 13 1 4 44 9 11 1 0 21 3 3 61 10 16 2 3 24 4 0 51 Total 230 16 21 204 .21 25 483 Student's t-test Chi-square test s2 = 67.278 yL2 = 178.256 t = 7.143 < 0.001 P = < 0.001 192 Appendix V.C.5. Fig. 38.e. Distribution of 192 hours old virgin female P. redivivus on agar strips after 6 hours at 25°C in response to males Number of worms per zone Replicate (E)x3 x2 xl 0 yl y2 y3 (E .100 ) 1 19 4 1 14 3 2 57 2 29 0 4 16 1 2 48 3 34 2 3 12 0 0 49 4 24 2 0 37 5 4 28 5 23 4 4 37 0 1 31 6 21 0 2 20 0 1 56 7 22 0 0 9 1 0 68 8 32 3 2 14 0 4 45 9 16 3. 2 41 0 2 36 10 10 2 2 29 7 - 6 44 Total 230 20 20 229 17 22 462 Student's t-test Chi-square test _ 2 2 s = 99.717 11(.. = 129.039 t = 5.173 < 0.001 < 0.001 193 Appendix V.C.5. Fig. 38.f. Distribution of 240 hours old virgin female P;'redivivus on agar strips after 6 hours at 259C in response to =le's- Number of worms per zone Replicate (E)x3 xl 0 yl y2 y3(E.100cr ) 1 20 0 0 32 0 1 47 2 18 3 3 18 3 3 52 3 22 2 1 22 0 49 4 17 2 1 38 4 0 38 5 20 3 1 36 2 1 37 6 24 0 0 18 2 2 54 7 10 4 8 37 2 7 32 8 33 3 3 12 1 0 48 9 15 3 2 54 0 3 23 10 13 3 3 45 0 1 35 Total 192 23 22 312 14 22 415 Student's t-test Chi-square test 2 .3(.2 s = 67.833 = 70.749 t = 5.810 P = < 0.001 P < 0.001 194 Appendix VI.A.3. Fig. 40. Effect of density of males on the timing (minutes after final moult) of the first copulation of female P. redivivus Number of male present Replicate 1 5 10 , 15 1 10 35 20 0.0 2 65 45 10 0.0 3 70 30 10 0.0 4 5 30 10 0.0 5 5 35 20 0.0 Total 225 175 70 0.0 Mean 45 35 14 0.0 S.D. 34.46 6.12 5.48 Regression line was drawn and significance of slope was compared. a 39.17 y = a a x o = o l a 1 = -2.47 = 39.17 - 2.47x 2 r 0.39 t = -2.47 0.73 Sy.x = 17.10 = 3.384 So = 6.81 < 0.01 S = 0.73 1 n = 20 df = 18 195 Appendix VI.B.3. Fig. 41 The effect of ageing of the'virgin female P. redivivus on the percentage of female copulating for the first time Hours old after final moult Replicate . 48 96 .144 192 240 1 10 9 9 8 8 2 10 10 9 9 10 3 8 9 10 10 8 Total 28 28 28 27 26 % copulated 93.3 93.3 93.3 90.0 86.6 S.D. 11.5 5.8 5.8 10.0 11.5 Regression line was drawn and significance of slope was compared a = o 96.33 al = -0.03 y = ao aix 2 r = 0.09 = 96.33 - 0.03x Sy.x = 8.27 t = -0.03 0.03 s = 5.01 o = 1.00 s = 0.03 1 P = > 0.1 = 15 df = 13 196 Appendix VI.B.3. Fig. 41 The effect of ageing of the virgin female P. redivivus on the number of eggs produced in the following 24 hours of copulation Hours old after final moult Replicate 48 96 144 192 240 1 27 26 40 31 10 2 25 29 35 34 18 3 32 35 39 24 19 4 35 27 35 37 12 5 22 18 30 29 00 6 13 27 35 38 10 7 18 35 25 31 15 8 25 27 33 29 12 9 16 24 19 46 17 10 21 26 15 32 7 11 24 33 29 23 10 12 27 24 15 25 00 13 21 21 25 34 14 14 39 19 23 19 00 15 18 35 31 41 5 16 16 25 21 39 00 17 18 28 27 36 8 18 10 34 22 33 00 19 11 23 21 - 24 20 21 22 26 - 00 21 27 28 20 - 00 22 19 32 30 - 7 23 23 39 17 - 00 24 25 31 31 - 8 25 17 32 16 - 15 26 21 37 15 - 21 27 22 36 21 28 28 33 24 29 - - 23 - - Total 621 806 743 581 232 Mean 22.2 28.8 25.6 32.3 8.9 S.D. 6.7 5.7 7.3 6.8 7.5 Student's t-test between egg production after 48 hours versus 192 hours 192 hours versus 240 hours df = 44 df = 42 2 2 s = 45.402727 s = 52.368 t = -4.96 t = 10.5 P = < 0.001 P = < 0.001 197 Appendix VI.C.3 a 4. b Fig. 42 The effect of increasing interval after the first copulation of the female P. redivivus on the % of the females copulating for second time with 2 - 3 hours isolated males Hours after first copulation Replicate 8 12 16 20 24 28 1 1 2 3 2 8 9 9 2 1 0 3 3 4 7 9 0 2 1 2 4 7 8 Total 2 4 7 7 16 23 26 % copulated 6.7 13.3 23.3 23.3 53.3 76.7 86.7 S.D. 5.8 .11.5 11.5 5.8 23.1 11.5 5.8 Fig. 42 The effect of the activity of the 12 4 hours isolated males on the % of the female P. redivivus copulating for the_second time with increasing intervals after the first copulation Hours after first copulation Replicate 4 8 12 16 20 24 28 1 0 0 3 7 9 10 10 2 0 3 2 5 7 8 9 3 0 3 1 6 7 10 10 Total 0 6 6 18 23 28 29 % copulated 0 20 20 60 76.6 93.3 96.7 S.D. - 17.3 10.0 10.0 11.5 11.5 5.8 198 Appendix VI.C.3 a + b Fig. 42 The effect of the higher number of the sperm received at the first copulation of the female P. redivivus on the % of the females copulating for the second time with increasing intervals after the first copulation Hours after first copulation Replicate 4 8 12 16 20 24 28 32 36 40 1 0 1 0 0 1 4 6 7 9 2 0 0 0 1 2 2 5 6 8 3 0 0 0 0 2 3 4 5 8 Total 0 0 1 1 4 6 13 17 23 27 % copulated 0 0 3.3 3.3 13.3 20.0 43.3 56.7 76.7 90.0 S.D. - 5.8 5.8 11.5 10.0 5.8 5.8 5.0 Above' three factors were compared by Friedman two-way analysis of variance by ranks before 12 hours after 12 hours 2 2 = 4.5 = 6 df = 2 df . = 2 P = > 0.1 P = <.0.05 199 Appendix VI.C.3a. Fig. 43 The % of female P. redivivus copulating for a second time with sperm from the first copulation(of 20 - 30 sperm)at varying time intervals from the first copulation Hours after first copulation Replicate 4 8 12 16 20 24 1 0 0 . 1 1 0 1 2 0 2 0 1 1 0 3 0 1 0 3 0 0 Total 0 3 1 5 1 1 % copulated 0 10 3.3 16.7 3.3 3.3 The % of female P. redivivus copulating for a second time without sperm from the first copulation(of 20 - 30 sperm) at varying time intervals from the first copulation Hours after first copulation Replicate 4 8 12 16 20 24 .1 0 2 6 9 9 2 0 2 4 6 7 3 0 2 1 3 7 10 Total 0 3 5 13 22 26 % copulated 0 10 16.7 43.3 73.3 86.7 200 Appendix VI.C.3b. Fig. 43. The % of female P. redivivus copulating for a second time with sperm from the firbt copulatioh (of 80 = 110 sperm)at varying time intervals from the first copulation Hours after first copulation Replicate 4 8 12 16 20 24 28 32 36 40 1 0 0 1 0 0 1 2 4 4 4 - 2 0 0 0 1. 2 2 5 6 6 4 3 0 0 0 0 2 3 4 5 6 3 Total 0 0 1 1 4 6 11 15 16 11 7. copulated 0 0 3.3 3.3 13.3 20 36.7 50.0 53.0 36.7 The % of female P. redivivus copulating for a second time without sperm from the first copulation(of 80-110 sperm)at varying time intervals from the first copulation Hours after first copulation Replicate 4 8 12 16 20 24 28 32 36 40 1 0 0 0 0 0 0 2 2 3 5 2 0 0 0 0 0 0 0 0 2 5 3 0 0 0 0 0 0 0 0 2 6 Total 0 0 0 0 0 0 2 2 7 16 % copulated 0 0 0 0 0 0 6.7 6.7 23.3 53.3 201 Appendix VI.C.3c. Fig.44 The number of oocytes fertilized (larvae stored) with increasing time interval after the first copulation with 20 - 30 sperm in newly moulted female P. redivivus Hours after first copulation Replicate 4 8 12 16 20 24 1 4 8 11 16 .22 5 2 4 6 10 19 18 9 3 5 6 8 15 23 22 4 4 9 10 26 19 18 5 4 4 11 16 20 17 6 4 5 .9 16 20 23 7 3 7 11 26 19 13 8 4 9 11 25 21 22 9 3 8 10 19 26 21 10 3 5 10 17 21 10 11 8 15 15 22 31 23 12 4. 13 18 23 19 25 13 4 13 20 23 18 34 14 7 12 17 15 25 21 15 5 15 16 19 21 29 16 4 12 18 23 28 26 17 5 11 17 20 20 19 18 4 13 17 18 23 24 19 6 9 21 21 31 25 20 6 10 23 25 26 20 21 4 10 19 21 35 23 22 7 15 17 25 32 21 23 6 15 21 24 24 31 24 6 10 22 22 29 31 25 4 6 18 14 22 21 26 9 14 23 29 31 29 27 5 14 18 20 37 29 28 5 17 - 24 26 28 17 29 4 11 21 25 23 31 30 - 8 13 20 24 31 31 Total 149 315 486 634 740 670 Mean 4.97 10.5 16.2 21.13 24.67 22.3 S.D. 1.56 3.59 4.88 4.02 5.51 6.99 202 Appendix VI.C.3b. Fig. 44 The number of oocytes fertilized (larvae stored) with increasing time interval after the first copulation with 80 - 110 sperm in newly moulted female P. redivivus Hours after first copulation Replicate 4 8 12 16 20 24 1 6 10 18 15 21 28 2 7 12 12 12 16 21 3 6 7 11 12 26 20 4 6 9 14 8 21 29 5 3 8 8 21 20 23 6 3 8 10 8 28 22 7 5 8 8 22 23 26 8 4 8 10 15 23 27 9 7 11 12 11 14 31 10 6 12 12 17 27 30 11 4 15 18 28 35 29 12 3 12 11 30 26 25 , 13 4 13 16 37 25 18 14 4 9 18 22 15 20 15 7 9 9 24 25 23 16 5 8 12 25 21 26 17 4 8 11 23 23 28 18 6 8 12 25 28 25 19 5 13 16 31 27 19 20 6 7 12 16 24 31 21 5 9 12 31 28 27 22 5 7 22 16 27 33 23 4 7 14 20 28 25 24 5 15 13 28 35 29 25 6 12 14 14 26 31 26 5 14 10 26 25 24 27 8 9 12 18 18 26 28 7 10 16 22 25 21 29 3 11 13 37 26 25 30 5 7 12 39 29 16 Total 154 296 388 653 735 758 Mean 5.13 9.87 12.93 21.77 24.5 25.27 S.D. 1.36 2.49 3.22 8.44 4.9 4.31 203 Appendix VI.C.3b. Fig.44 The number of oocytes fertilized (larvae stored) with increasing time interval after the first copulation with 20 - 30 sperm in 48 hour old virgin female P. redivivus Hours after first copulation Replicate 4 8 12 16 20 24 1 8 21 15 37 27 25 2 . 8 25 16 25 25 28 3 6 19 26 23 21 39 4 10. 21 32 33 37 34 5 13 26 10 23 31 39 6 10 21 19 29 35 30 7 10 17 26 25 44 35 8 7 19 18 41 46 39 9 5 18 24 28 23 29 10 12 23 35 20 36 33 11 7 29 37 37 34 21 12 8 27 25 20 41 19 13 7 26 23 25 31 26 14 9 21 33 36 27 26 15 11 20 23 34 25 27 16 12 18 29 34 26 41 17 11 19 25 27 27 35 18 8 28 41 33 35 21 19 11 19 28 16 20 28 20 10 24 20 35 21 29 21 7 17 33 37 25 26 22 7 16 33 26 33 35 23 11 15 20 24 28 26 24 12 33 25 34 29 28 25 14 21 36 43 39 26 26 13 21 18 45 39 28 27 17 22 47 38 34 29 28 .15 18 30 29 35 31 29 8 25 15 27 39 25 30 10 16 31 36 30 27 Total 297 645 793 920 943 885 Mean 9.9 21.5 26.43 30.7 31.43 29.5 S.D. 2.82 4.33 8.45 7.24 6.93 5.59 204 Appendix VI.C.3b. Fig. 44 The number of oocytes fertilized (larvae stored) with increasing time interval after the first copulation with 20 - 30 sperm in 96 hour old virgin female P. redivivus Hours after first copulation Replicate 4 8 12 16 20 24 1 13 15 17 21 17 33 2 11 23 21 18 21 18 3 10 28 21 25 18 20 4 14 16 24 25 27 34 5 13 17 15 20 13 28 6 7 18 23 30 32 31 7 21 21 22 31 36 25 8 15 20 16 25 31 29 9 14 16 18 18 29 32 10 15 19 22 14 15 31 11 16 19 30 27 29 29 12 10 28 21 29 41 32 13 20 26 14 27 35 18 14 16 17 27 18 17 22 15 13 25 24 29 30 42 16 14 19 17 23 10 29 17 15 17 40 15 30 43 18 18 18 26 19 31 29 19 13 23 23 25 40 35 20 9 23 25 17 27 34 21 20 29 24 20 27 34 22 14 24 15 22 32 41 23 8 19 26 28 18 31 24 10 17 17 25 27 27 25 18 22 25 17 24 26 26 9 29 22 21 29 25 27 17 17 27 19 26 27 28 11 15 30 18 25 35 29 19 21 16 23 38 20 30 8 18 12 28 26 21 Total 411 619 660 677 801 881 Mean 13.7 20.63 22.0 22.57 26.7 29.37 S.D. 3.88 4.28 5.86 4.76 7.84 6.61 205 Appendix VI.C.3c. Fig. 46 The effect of 48 hour virginity of the female P. redivivus on ' the % of the females copulating for the second time with increasing intervals after the first copulation with 20-30 sperm Hours after first copulation Replicate 4 8 12 16 20 24 1 4 7 8 9 10 2 1 3 8, 8 9 -9 3 2 4 6 9 10 10 Total copulated 4 11 21 25 28 29 % copulated 13.3 36.7 70.0. 83.3 93.3 96.7 S.D. 5.8 5.8 10.0 5.8 5.8 5.8 The effect of 96 hour virginity of the female P. redivivus on the % of the females copulating for the second time with increasing intervals after the first copulation with 20-30 sperm Hours after first copulation Replicate 4 8 12 16 20 24 1 1 3 7 8 8 8 2 2 2 6 6 7 10 3 3 2 5 7 8 9 Total 6 7 18 21 23 27 % copulated 20.0 23.3 60.0 70.0 76.6 90.0 S.D. 10.0 5.8 10.0 10.0 5.8 10.0 The above two sets of data was compared with that of newly moulted copulated female by Friedman two-way analysis of variance by ranks. 2 = 7.58 df = 2 P = < 0.05 206 Appendix VI.D.2. Fig. 47 The % of female P. redivivus copulating for a third time with sperm from the second copulation(of 20 - 30 sperm)at varying time intervals from the previous copulation Hours after second copulation Replicate 4 8 12 16 20 24 1 1 2 2 0 1 2 2 1 1 3 1 1 1 3 2. 2 1 0 0 5 Total 4 5 6 1 2 8 % copulated 13.3 16.7 20.0 3.3 6.7 26.7 The % of female P. redivivus copulating for a third time without sperm from the second copulation(of 20 - 30 sperlOat varying time intervals from the previous copulation Hours after second copulation Replicate 4 8 12 16 20 24 1 0 1 2 5 8 6 2 0 0 1 5 .7 9 3 0 1 2 5 8 4 Total 0 2 5 15 23 19 % copulated 0 6.7 16.7 50.0 76.7 63.3