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The effect of the age of female Nasonia vitripennis (Walker) (Hymenoptera, Pteromalidae) upon the incidence of larval diapause
J. Ins. Pkysiol., 1962, ?‘aZ. 8, pp. 309 to 318. Pergamon Press Ltd. Printed in Great &&in
THE EFFECT OF THE AGE OF FEMALE NASONIA V_lTRIPENNIS (WALKER) (HYMENOPTERA, PTEROMALIDAE) UPON THE INCIDENCE OF LARVAL DIAPAUSE D. S. SAUNDERS Department of Zoology, University of Edinburgh (Received 23 December 1961)
Abstract-A study of the incidence of diapause in the progeny of single females of Nasonia vitripennis has been made throughout their lives. Although individual results are variable, it is shown that as the maternal generation becomes more senile, a greater proportion of the progeny enter diapause at the end of the fourth larval instar. In many females, the change-over from the production of non-diapause to diapause offspring is extremely sudden. A few females are shown to have the opposite trend, producing more diapause larvae at the beginning of life. An overall comparison of the observed and expected frequencies of diapause in the first and second halves of the life-span, however, is highly significant.
INTRODUCTION
THE pteromalid Nusonia vitripmnis (Walk.) is a common ectoparasite of Muscoid pupae which enters a facultative diapause at the end of the fourth and final larval instar. Much has been written about the biology of this chalcid (ROUBAUD,1917; ALTSON, 1920; COUSIN, 1933; EVANS, 1933; VAN DER MERWE, 1943; EDWARDS, 1954, etc.); and its development and diapause have been recently described in detail by SCHNEIDERMAN and HORWITZ(1958). According to these authors, a larva which completes its development empties its gut at the end of the fourth instar, becomes a prepupa, and finally moults to the pupal stage, without interruption. A diapause larva, on the other hand, fails to defaecate and may remain unchanged for up to several years in the laboratory unless diapause is broken by exposure to low temperature. Th e signs that deveIopment has recommenced are defaecation and the pupal moult. Several attempts have been made to establish the factors which induce diapause in N. vitripennis. According to COUSIN (1932), deficient nutrition, as a result of superparasitism, caused the larvae to enter diapause. VAN DER MERWE (1943) claimed that low humidity or falling temperature acting upon the chalcid larvae within the host puparium were also responsible. The most comprehensive study of diapause in this insect is by SCHNEIDERMAN and HORWITZ(1958). These authors found that factors acting upon the larvae within the host-such as superparasitism, 309
310
D. S.
SAUNDERS
the age or condition of the host pupa, humidity, temperature, and photoperiodhad no effect, but chilling the female parent during oiigenesis, or depriving her of hosts from which the parasite normally acquires protein exudates which further egg production (ROUBAUD,1917), caused her offspring to enter diapause at the end of the fourth instar. In another pteromalid, Spalangia drosophilae, SIMMONDS (1946, 1948) sh owed that the age of the female parent also had some effect upon the incidence of diapause in her offspring, a greater proportion of the progeny of more senile females ceasing development. SCHNEIDERMAN and HORWITZ (1958) investigated the effect of the senility of N. vitripennis on the induction of diapause but came to the conclusion that there was no connexion between these two variables. The work reported in the present paper was done between 1956 and 1958, before the publication of SCHNEIDERMAN and HORWITZ’Spaper, and is presented as evidence that, at least in the strain of N. vitripennis used, the age of the female parent does have an effect upon the incidence of diapause in her offspring. The possibility of strain differences being the reason for the discrepancy between the present results and those of SCHNEIDERMAN and HORWITZis considered in the discussion. MATERIALS AND METHODS 1. Culture methods The strain of NusonM vitripennis was obtained in London from a naturally infested culture of Lucilia sericata. It was subsequently bred in puparia of Lucilia at 2526°C at about 70 per cent r.h. The adult chalcids were kept in 10 in. cubical cages of Perspex and Nylon gauze and supplied with glucose syrup and fresh host pupae daily. Parasitized hosts were removed from the cage after 3 hr, enclosed in 3 x 1 in. glass tubes and incubated at 25°C and 60 per cent r.h. At emergence, adult chalcids were used to restock the culture cages. Host Lucilia sericata were reared as adults on meat, sugar, and water, and as larvae on a mixture of yeast, agar, and dried milk in biscuit tins. The larvae were allowed to pupate on the floor of these tins without the presence of sawdust, so that pupae of known age could be collected daily. Pupae offered to Nusoniu for parasitization were all 2-day-old pupae in which the subpuparial space (in which the chalcid larvae live and feed) was fully formed, i.e. puparia containing newly formed pupae. 2. Experimental method A batch of chalcid pupae, all progeny of female N. vitripermis bred at 2526”C, were dissected out from host Lucilia. Twenty females which emerged from these pupae were enclosed singly in small glass tubes together with a male and a drop of glucose syrup, and incubated at 25°C and 60 per cent r.h. A 2-day-old pupa of Luslia was then added daily to each tube, the previous day’s pupa being removed and incubated separately at the same conditions of temperature and humidity as the parent generation. At emergence, the progeny resulting from each of these hosts were recorded. The pupae were then left for several days and dissected to recover the diapause larvae, which were easily recognized by the semi-transparent nature of the fat-body (VANDERMERLE, 1943), the presence of faecal material in the midgut, and the fact that, although in most cases considerable host material remained, the larvae had not completed their development along with the adult chalcids which emerged from the same host. This procedure was continued with the progeny of each female Nasonia until her death.
EFFECT OF AGE OF NASONIA
VITRIPENNIS
UPON
LARVAL
311
DIAPAUSE
RESULTS
Although the twenty female Nasonia used in the experiment were all progeny of females drawn from a population breeding in a controlled environment, there was a great variability in the proportions of their offspring entering diapause (Table 1). Of the twenty females, one died on the second day of the experiment TABLE
I-THE
NUMBER
OF DEVELOPING
AND
DIAPAUSING
OFFSPRING
OF NINETEEN
FEMALE
Nasonia vitripennis Total no. offspring
Total no. adults
Males
Females
144 290 195 67 126 61 135 178 186 121 112 118 172 35 5 0 0
14 41 35 14 37 18 36 20 23 20 39 45 41 10 2 0 0
130 249 160 53 89 43 99 158 163 101 73 73 131 25 3 0 0
4 5 4 12 10 39 56 71 65 64 72 191 127 245 195 130
390 84
0 0
0 0
No. diapause larvae
% diapause larvae
(a) Fertilized females 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1.5 16 17
144 294 200 71 138 71 174 234 257 186 176 190 363 162 250 195 130
0
0.00 1.36 2.50 5.63 8.69 14.08 22.41 23.93 27.63 34.95 36.36 37.89 52.62 78.39 98.00 100~00 100~00
(b) Unfertilized females 18 19
390 84
390 84
0.00 0.00
Each line is concerned with a single female.
and has been eliminated from the results; two produced only male progeny (all of which had an uninterrupted development) and were presumably unfertilized ; and the other seventeen produced both diapausing and non-diapausing offspring sometime during their life. The number and proportion of diapause larvae, however, were extremely variable and included one female (No. 1) which produced none of her offspring as diapausing individuals and, at the other extreme, two (16 and 17) with all their progeny in diapause. These variable results are very similar to those obtained by SIMMONDS (1948) for Spalangia drosophilae.
;; 21 22 23 24 25 26 27 28 29
15 16 17 18
12 13 14
(days)
Age of female
TABLE
2-A
0
0
0
0 0 0 0 0
0
Died
0
-
0 0 5 (23.8) 0
0 0
Dyed
: (100)
0
4
0 0 0 0
RECORD
9 (52.8) 0 D!ed
01 (5.2) -
6
NUMBER
to produce
OF THE
with tendency
3
Females
CHRONOLOGICAL
AND
PERCENTAGE OF PROGENY FEMALES
ENTERING
21 (100) Died
1: (100) -
: 0 0 0 0 0 0
0 0 0
: 0
7
(12.5) (100) (100) (100)
2(100) Died
: 0 1 8 15 21 -
0 0
: 0 0 0 0 0 0 0
:
9 (50) 0
8
-
Died
-
19 (100) 14 (100) 19 (95)
1:(100)
2 (12.4) 0 0 0
0 0
0 0
: 0 0 0
0 0
0
:
9
an increased number of diapause offspring Female No.
Vit&??WliS
Died
-
15 (100) -
14 (100) 18 (100)
1:(100)
: 0 0 0
:
ii Died
0 0
(100)
1;
18 (100) -
-
1: (100) 14 (100) -
7 (50)
i
:
0
0 ii
11
the end of life
IN SEVENTEEN
10
towards
DIAPAUSE
25(100) 22 (95.6) 15 (100) Died
22(100) 19 (100) 20 (100) 23 (100)
22(100)
23 (100) -
13
Nasonia N
2
8
: 0
0 -
-0
0
: 7
z 10
:;
13 14 :;
23
ii
20
17 :t
0
;:
-
Died
0
:
0 0 0
-:
1: (25)
2
Died
-
: (40.9) 0 1 (7.7) 0
:
:
lK(100) 29 (100) 0
1: (100)
12
-
Died
00
0
:
0
0
1
Low level diapause Female No.
0
14 (87.5) Died
19 (GO) 19 (100) -
7
17 (GO, -
8 (38.1) 18 (100) 18 (100)
2 (28.5) (loo, (100) (100) (100)
20 (loo) 18 (100) Died
17 (loo> 18 (100) -
15 (loo, -
17 (loo) 14 (73.7) 19 (100) 1.5 (100) -
18 (GO) -
11 16 18 20
9 (GO,
15
(100) (100) (100) (100) (100) -
llD(Y~)
11 22 (100)
18 19 20 15 10
15
22 20 (100)
14 (100) Died
16(100)
18(1000)
12
17
16
producing a consistently high proportion of diapause larvae Female No.
5 (1W
14
Females
The horizontal lines indicate the mid-point in each life-span. The figures in brackets indicate the percentage of the brood in diapause.
: Died
:: 0
: 0 0 0
1: (100)
5
Females producing more diapause larvae at the beginning of life Female No.
::
Age of female (days)
8 % % g
k
E
S 2
s 2;
? FE
r;:
$
3
0
!I $
314
D.
S. SAUNDERS
An analysis of the effect of ageing upon the progeny of single females is shown in Table 2. Four of the seventeen fertilized Nasonia (Nos. 14 to 17) produced a high proportion of diapause larvae throughout life, and one (No. 1) produced all its offspring as non-diapausing individuals. The remaining twelve females were of two types: nine showed a tendency to produce more diapause offspring at the end of life and the other three tended to produce more diapause larvae at the beginning of life. If the results for the seventeen females are combined (Table 3), an overall TABLE ~-SHOWING
THE EFFECTOF THE AGE OF FEMALE Nasonia vitripe-nnis UPON THE PERCENTAGEOF THE PROGENY ENTERING DIAPAUSE
(days)
No. of females alive
1 2 5 6 7 8 9 11 12 13 14 15 16 18 19 20 21 22 23 24 25 26 27 28
17 17 17 17 17 16 15 15 15 12 12 12 12 12 10 7 7 6 6 6 5 3 3 1
Age of females
Total :;a spring 32 222 231 329 312 173 298 55 173 93 85 178 128 46 167 69 91 89 89 29 20 43 25 15
Total no. adults
No. of diapause larvae
% diapause larvae
Log. % diapause larvae
27 155 178 245 236 130 214 37 89 45 32 94 68 20 88 28 25 23 29 6 0 0 1 0
5 67 53 84 76 43 84 18 84 48 53 84 60 26 79 41 66 66 60 23 20 43 24 15
IS.62 30.18 22.94 25.53 24.36 24.85 28.19 32.73 48.55 24.87 62.35 47.19 46.87 56.52 47.30 59.42 72.53 74.16 67.41 79.31 100~00 100~00 96.00 100~00
I.1937 I.4797 I.3606 I.4070 I.3867 1.3954 I.4501 I.5149 I.6861 I.3957 I.7948 I.6738 I.6709 I.7522 I.6749 I.7739 I.8605 I.8702 I.8288 1.8994 2.0000 2.0000 I.9823 2.0000
increase in the proportion of the progeny entering diapause is shown to occur as the parent females become more senile. Plotting the age of the females against the logarithm of percentage diapause (Fig. 1) results in a straight line. These results show that, for the female Nasonia investigated, there is a positive association between the age of the females at oviposition and the proportion of the resultant It is pointed out, however, that the number of females larvae entering diapause.
EFFRCT
OF AGE
OF NASONIA
V~T~P~~NIS
UPON LARVAL
315
DlAPAUSR
alive on any one day drops during the experiment from seventeen to one. This means that the reliability of the results for the older females is less than that for the younger females. . .
2.0-
.
.-
1.2-* 0
I 5
I I 15 20 IO Age of maternal generation,
25
30
days
FIG. 1. The effect of the age of the maternal generation of Nasonia oitripmnis the proportion of the progeny entering diapause.
upon
In view of the heterogeneity of the results, they were tested for significance by the following method. The life-span of each female was divided into two to give a ‘young’ half and an ‘old’ half. Knowing the overall percentage of the offspring of each female entering diapause, it was then possible to calculate the expected frequency of diapause larvae in each half life-span, assuming an even distribution between the two halves. These expected values were then compared with the observed rates of diapause and a series of x2 worked out (Table 4). These were combined by summing the x’s, dividing by ,/17 and finally squaring the figure obtained. In the case of those females which showed a drop in the proportion of progeny entering diapause as they became older, the x obtained was treated as a negative value in the summation. This procedure gave a combined xa of 95.96 (P ~O*OOl) and shows that the tendency to produce more diapause larvae in the second half of life is highly significant. DISCUSSION Several
examples of diapause being induced by factors affecting the maternal generation are now known. SIMMONDS(1946, 1948) showed that old females of Spalangia drosophilae, a pteromalid parasite of the fritfly, produced a greater proportion of diapause larvae in their offspring than young females. The results
D.
316 shown
in the present
diapause working
occurs
paper
in ?&.so&z
with N. ~~~~~n~,
oiigenesis,
or depriving
S. SAUNDERS
are evidence
that a similar
~~~~~~~~. found
them
relation
that exposing
of hosts
females
in which
between
and HORW~TZ
~CHNE~D~N
age and
(1958),
to low temperature
to oviposit,
also
also
during
induced
larval
TABLE ~-THE OBSERVEDAND EXPECTED FREQUENCIESOF DIAPAUSE LARVAE IN THE OFFSPRING OF FEMALE Nasonia VitYi@?lniS,SHOWING THAT THE GREATER PROPORTION OF DIAPAUSE LARVAE ARE PRODUCED BY A FEMALE IN THE SECOND HALF OF LIFE
Total no. in diapause
Total RO. Off-
% diapause of total
spring 1 2 3 4 5 ! 8 9 10 11 12 13 14 15 16 17
144 294 200 71 138 71 174 234 257 186 176 190 363 162 250 195 130
040 1.36 2.50 5.63 8.69 14.08 22.41 23‘93 27.63 34.95 36.34 37.89 52.62 78.39 98.00
0 4 5 4 12 10 39 56 71 65 64 72 191 127 245 195 130
100.00 100*00
No. of diapause larvae Observed Expected Xs
*
t
*
t
0 4 0 0 12 1 0 9 0 0 34 62 0 68 123 69 53
0 0 5 4 0 9 39 47 71 65 30 10 191 59 122 126 77
0 l-73 3.35 0.51 6.26 2.67 25.32 2740 32.60 35.99 37.88 43.57 85.00 79.17 125.41 69.00 53.00
0 2.27 1.6s 3.49 5.74 7.32 13.67 28.59 38.40 29.01 26.11 28-42 105.99 47.82 117.57 126.00 77.00
X
0.~ - 2.2910 3.2410 0.2821 - 3.3170 1.2830 8.4980 4.9190 7.7630 8.9440 0.9866 - 4*44-M 12.3800 2+wo O-0980 0~0000 0~0000
0.~0 5.2480 10.1492 0.0796 1 lGo20 1.6479 72.2185 24.1981 60.2760 80.~ 0.9739 19.7474 153.2100 4.1850 0.~96 o*oooo 0~0000
CX = 40.3877.
(pg =f (4Eg)” = 9.79572 = 95.96,
P 4 O*OOl.
* First haif of life-span. t Second haIf of life-span. diapause
in the
influence
on the induction
ser&-&.z
offspring.
the
Chalcidoidea,
hormone
been
shown
produced
silkworm, generation
to enter diapause
however, is most
(FUKUDA,
by the sub-oesophageal
causes the progeny hormone
in the silkworm
the stimulus
effective
(KOGURE,
to the eggs
1933) ; the stimulus
by almost a generation
1951;
HASEGAWA,
in females the egg.
which
initiates give
‘and the resultant
in this insect.
a maternal by L&&z
(ROUBAU~,
within which
of
are provided
~u~~~~~
ganglion
as embryos
(illumination)
if applied
examples
in the offspring
(CRAGG and COLE, 1952) and ~~~e~o~~~~
has also
separated
Outside
of diapause
1928).
1951)
during
It
that a
oijgenesis
In the case of the the release
of the
rise to the maternal
diapause
are therefore
EFF%CTOF AGE OF NASONM
Z?TRIPENNIS
UPON LARVAL DIAPAUSE
317
In Naso?& nitripennti, the mechanisms by which these maternal influences operate is not known. It is apparent, however, that females exposed to low temperature, or to the effects of age or host deprivation during o6genesis, must lay eggs which, because they give rise to diapausing progeny, differ in some way from normal eggs. If diapause in the larvae of NU~O&Zis under some sort of humoral control, as in several other insects (see LED, 1955), these maternal influences must affect the hormone balance of the larva by some mechanism which is transmitted through the ovary. SCHNEIDERMAN and HORW~TZpoint out that low temperature could act directly upon the ovaries during oiigenesis and result in the production of ‘diapause’ eggs, or through the mediation of another organ as in the silkworm. The same could also be said of the effect of the age of the female parent and of host deprivation. N~~~~ w~~~~~~~ occurs in several different continents and climates, and there is evidence that strain differences occur within the species. For instance, COUSIN(1933) working with N. vitripennis from France, SAUNDERS (1958) with London material, and SCHNEIDERMAN and HORWITZ (1958) working with a strain obtained from Woods Hole all record the duration of the parasitic phase of the life cycle as 14 days at 25°C. Vhr~ DER MERWE (1943), on the other hand, working in South Africa, records a slightly longer developmental period at this temperature. Furthermore, SCHNEIDERMANand HORWITZ found that chilling the female parent induced diapause in the offspring, but quote MOURSI(1946) who failed to induce diapause by this method in Californian N. vitripennnis. It is probable, therefore, that the discrepancy between the present results obtained with the London material and those of SC~NEIDERMAN and HORWITZwith the Woods Hole material is also due to differences in strain. Acknowledgements-This work was carried out in the Department of Entomology, London School of Hygiene and Tropical Medicine, whilst the author was in receipt of a Medical Research Council Scholarship. The author is grateful to Professor D. S. BERTRAM for his help during the investigation and to Dr. BRYAN CLARKE for discussing the statistical treatment of the results. REFERENCES ALTSON A. M. (1920) The life history and habits of two parasites of blowflies.
Proc. 2001.
Sot. Lond. 15, 196-243. COUSIN G. (1932) Etude experimentale de la diapause des insectes. Bull. biol. (Suppi.) 15, l-341. COUSIN G. (1933) Etude biologique d’un chalcidien: Illormortie~~aaitripennis Walk. Bull.
biol. 67, 371400. CRAGGJ. B. and COLE P. (1952) Diapause in Luciliu swicata (Mg.) Diptera. r. exp. Biol. 29, 600-604. EDWARDS R. L. (1954)
The host-finding and oviposition behaviour of ~o~on~eZla n&pipennis (Walker) (Hym. Pteromalidae), a parasite of Muscoid flies. Behaviour 7, 88-112. EVANS A. C. (1933) Comparative observations on the morphology and biology of some Hymenopterous parasites of carrion-infesting Diptera. Bull. ent. Res. 24, 385-405. FUKUDA S. (1951) Production ganglion in the silkworm.
of the diapause eggs by transplanting
Proc. imp. Acad.Japan
27, 672-677.
the subesophageal
318
D. S. SAUNDERS
HASEGAWAK. (1951) Studies in voltinism in the silkworm, Bombyx mori L., with special reference to the organs concerning determination of voltinism (A preliminary note). Proc. imp. Acad. Japan 27, 667-671. KOGURE M. (1933) The influence of light and temperature on certain characters of the silkworm, Bombyx mori. J. Dep. Agric. Kyushu Univ. 4, l-93. LEES A. D. (1955) The Physiology of Diapause in Arthropods. Cambridge University Press. MERWE J. S. VAN DER (1943) Investigations on the biology and ecology of Mormoniella vitripennis Walk. (Pteromalidae, Hym.). J. ent. Sot. S. Afr. 6, 48-64. MOUR~I A. A. (1946) The effect of temperature on development and reproduction of Mormoniella vitripennis (Walker). Bull. Sot. ent., l?gypte 30, 39-61. ROUBAUDE. (1917) Observations biologiques sur Nasonia brevicornis Ashm., Chalcidide parasite des pupes des Muscides. Detenninisme physiologique de l’instinct de ponte; adaptation a la lutte contre les glossines. Bull. sci. Fr. Belg. 1, 425-439. ROUBAUDE. (1928) Asthenobiose et hibernation obligatoire provoquee chez P. papatasii Stop. Bull. Sot. Pat. exot. 21, 436439. SAUNDERSD. S. (1958) The biology of parasites of fly puparia. Ph.D. thesis, University of London. SCHNEIDERMAN H. A. and HORWITZJ. (1958). The induction and termination of facultative diapause in the chalcid wasps Mormoniella vitripennis (Walker) and Tritneptis klugii (Ratzeburg). J. exp. Biol. 35, 520-551. SIMMONDS F. J. (1946) A factor affecting diapause in Hymenopterous parasites. Bull. ent. Res. 37, 95-97. SIMMONDS F. J. (1948) The influence of maternal physiology on the incidence of diapause. Phil. Trans. (B) 233, 385414.
THE EFFECT OF THE AGE OF FEMALE NASONIA V_lTRIPENNIS (WALKER) (HYMENOPTERA, PTEROMALIDAE) UPON THE INCIDENCE OF LARVAL DIAPAUSE D. S. SAUNDERS Department of Zoology, University of Edinburgh (Received 23 December 1961)
Abstract-A study of the incidence of diapause in the progeny of single females of Nasonia vitripennis has been made throughout their lives. Although individual results are variable, it is shown that as the maternal generation becomes more senile, a greater proportion of the progeny enter diapause at the end of the fourth larval instar. In many females, the change-over from the production of non-diapause to diapause offspring is extremely sudden. A few females are shown to have the opposite trend, producing more diapause larvae at the beginning of life. An overall comparison of the observed and expected frequencies of diapause in the first and second halves of the life-span, however, is highly significant.
INTRODUCTION
THE pteromalid Nusonia vitripmnis (Walk.) is a common ectoparasite of Muscoid pupae which enters a facultative diapause at the end of the fourth and final larval instar. Much has been written about the biology of this chalcid (ROUBAUD,1917; ALTSON, 1920; COUSIN, 1933; EVANS, 1933; VAN DER MERWE, 1943; EDWARDS, 1954, etc.); and its development and diapause have been recently described in detail by SCHNEIDERMAN and HORWITZ(1958). According to these authors, a larva which completes its development empties its gut at the end of the fourth instar, becomes a prepupa, and finally moults to the pupal stage, without interruption. A diapause larva, on the other hand, fails to defaecate and may remain unchanged for up to several years in the laboratory unless diapause is broken by exposure to low temperature. Th e signs that deveIopment has recommenced are defaecation and the pupal moult. Several attempts have been made to establish the factors which induce diapause in N. vitripennis. According to COUSIN (1932), deficient nutrition, as a result of superparasitism, caused the larvae to enter diapause. VAN DER MERWE (1943) claimed that low humidity or falling temperature acting upon the chalcid larvae within the host puparium were also responsible. The most comprehensive study of diapause in this insect is by SCHNEIDERMAN and HORWITZ(1958). These authors found that factors acting upon the larvae within the host-such as superparasitism, 309
310
D. S.
SAUNDERS
the age or condition of the host pupa, humidity, temperature, and photoperiodhad no effect, but chilling the female parent during oiigenesis, or depriving her of hosts from which the parasite normally acquires protein exudates which further egg production (ROUBAUD,1917), caused her offspring to enter diapause at the end of the fourth instar. In another pteromalid, Spalangia drosophilae, SIMMONDS (1946, 1948) sh owed that the age of the female parent also had some effect upon the incidence of diapause in her offspring, a greater proportion of the progeny of more senile females ceasing development. SCHNEIDERMAN and HORWITZ (1958) investigated the effect of the senility of N. vitripennis on the induction of diapause but came to the conclusion that there was no connexion between these two variables. The work reported in the present paper was done between 1956 and 1958, before the publication of SCHNEIDERMAN and HORWITZ’Spaper, and is presented as evidence that, at least in the strain of N. vitripennis used, the age of the female parent does have an effect upon the incidence of diapause in her offspring. The possibility of strain differences being the reason for the discrepancy between the present results and those of SCHNEIDERMAN and HORWITZis considered in the discussion. MATERIALS AND METHODS 1. Culture methods The strain of NusonM vitripennis was obtained in London from a naturally infested culture of Lucilia sericata. It was subsequently bred in puparia of Lucilia at 2526°C at about 70 per cent r.h. The adult chalcids were kept in 10 in. cubical cages of Perspex and Nylon gauze and supplied with glucose syrup and fresh host pupae daily. Parasitized hosts were removed from the cage after 3 hr, enclosed in 3 x 1 in. glass tubes and incubated at 25°C and 60 per cent r.h. At emergence, adult chalcids were used to restock the culture cages. Host Lucilia sericata were reared as adults on meat, sugar, and water, and as larvae on a mixture of yeast, agar, and dried milk in biscuit tins. The larvae were allowed to pupate on the floor of these tins without the presence of sawdust, so that pupae of known age could be collected daily. Pupae offered to Nusoniu for parasitization were all 2-day-old pupae in which the subpuparial space (in which the chalcid larvae live and feed) was fully formed, i.e. puparia containing newly formed pupae. 2. Experimental method A batch of chalcid pupae, all progeny of female N. vitripermis bred at 2526”C, were dissected out from host Lucilia. Twenty females which emerged from these pupae were enclosed singly in small glass tubes together with a male and a drop of glucose syrup, and incubated at 25°C and 60 per cent r.h. A 2-day-old pupa of Luslia was then added daily to each tube, the previous day’s pupa being removed and incubated separately at the same conditions of temperature and humidity as the parent generation. At emergence, the progeny resulting from each of these hosts were recorded. The pupae were then left for several days and dissected to recover the diapause larvae, which were easily recognized by the semi-transparent nature of the fat-body (VANDERMERLE, 1943), the presence of faecal material in the midgut, and the fact that, although in most cases considerable host material remained, the larvae had not completed their development along with the adult chalcids which emerged from the same host. This procedure was continued with the progeny of each female Nasonia until her death.
EFFECT OF AGE OF NASONIA
VITRIPENNIS
UPON
LARVAL
311
DIAPAUSE
RESULTS
Although the twenty female Nasonia used in the experiment were all progeny of females drawn from a population breeding in a controlled environment, there was a great variability in the proportions of their offspring entering diapause (Table 1). Of the twenty females, one died on the second day of the experiment TABLE
I-THE
NUMBER
OF DEVELOPING
AND
DIAPAUSING
OFFSPRING
OF NINETEEN
FEMALE
Nasonia vitripennis Total no. offspring
Total no. adults
Males
Females
144 290 195 67 126 61 135 178 186 121 112 118 172 35 5 0 0
14 41 35 14 37 18 36 20 23 20 39 45 41 10 2 0 0
130 249 160 53 89 43 99 158 163 101 73 73 131 25 3 0 0
4 5 4 12 10 39 56 71 65 64 72 191 127 245 195 130
390 84
0 0
0 0
No. diapause larvae
% diapause larvae
(a) Fertilized females 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1.5 16 17
144 294 200 71 138 71 174 234 257 186 176 190 363 162 250 195 130
0
0.00 1.36 2.50 5.63 8.69 14.08 22.41 23.93 27.63 34.95 36.36 37.89 52.62 78.39 98.00 100~00 100~00
(b) Unfertilized females 18 19
390 84
390 84
0.00 0.00
Each line is concerned with a single female.
and has been eliminated from the results; two produced only male progeny (all of which had an uninterrupted development) and were presumably unfertilized ; and the other seventeen produced both diapausing and non-diapausing offspring sometime during their life. The number and proportion of diapause larvae, however, were extremely variable and included one female (No. 1) which produced none of her offspring as diapausing individuals and, at the other extreme, two (16 and 17) with all their progeny in diapause. These variable results are very similar to those obtained by SIMMONDS (1948) for Spalangia drosophilae.
;; 21 22 23 24 25 26 27 28 29
15 16 17 18
12 13 14
(days)
Age of female
TABLE
2-A
0
0
0
0 0 0 0 0
0
Died
0
-
0 0 5 (23.8) 0
0 0
Dyed
: (100)
0
4
0 0 0 0
RECORD
9 (52.8) 0 D!ed
01 (5.2) -
6
NUMBER
to produce
OF THE
with tendency
3
Females
CHRONOLOGICAL
AND
PERCENTAGE OF PROGENY FEMALES
ENTERING
21 (100) Died
1: (100) -
: 0 0 0 0 0 0
0 0 0
: 0
7
(12.5) (100) (100) (100)
2(100) Died
: 0 1 8 15 21 -
0 0
: 0 0 0 0 0 0 0
:
9 (50) 0
8
-
Died
-
19 (100) 14 (100) 19 (95)
1:(100)
2 (12.4) 0 0 0
0 0
0 0
: 0 0 0
0 0
0
:
9
an increased number of diapause offspring Female No.
Vit&??WliS
Died
-
15 (100) -
14 (100) 18 (100)
1:(100)
: 0 0 0
:
ii Died
0 0
(100)
1;
18 (100) -
-
1: (100) 14 (100) -
7 (50)
i
:
0
0 ii
11
the end of life
IN SEVENTEEN
10
towards
DIAPAUSE
25(100) 22 (95.6) 15 (100) Died
22(100) 19 (100) 20 (100) 23 (100)
22(100)
23 (100) -
13
Nasonia N
2
8
: 0
0 -
-0
0
: 7
z 10
:;
13 14 :;
23
ii
20
17 :t
0
;:
-
Died
0
:
0 0 0
-:
1: (25)
2
Died
-
: (40.9) 0 1 (7.7) 0
:
:
lK(100) 29 (100) 0
1: (100)
12
-
Died
00
0
:
0
0
1
Low level diapause Female No.
0
14 (87.5) Died
19 (GO) 19 (100) -
7
17 (GO, -
8 (38.1) 18 (100) 18 (100)
2 (28.5) (loo, (100) (100) (100)
20 (loo) 18 (100) Died
17 (loo> 18 (100) -
15 (loo, -
17 (loo) 14 (73.7) 19 (100) 1.5 (100) -
18 (GO) -
11 16 18 20
9 (GO,
15
(100) (100) (100) (100) (100) -
llD(Y~)
11
18 19 20 15 10
15
22
14 (100) Died
16(100)
18(1000)
12
17
16
producing a consistently high proportion of diapause larvae Female No.
5 (1W
14
Females
The horizontal lines indicate the mid-point in each life-span. The figures in brackets indicate the percentage of the brood in diapause.
: Died
:: 0
: 0 0 0
1: (100)
5
Females producing more diapause larvae at the beginning of life Female No.
::
Age of female (days)
8 % % g
k
E
S 2
s 2;
? FE
r;:
$
3
0
!I $
314
D.
S. SAUNDERS
An analysis of the effect of ageing upon the progeny of single females is shown in Table 2. Four of the seventeen fertilized Nasonia (Nos. 14 to 17) produced a high proportion of diapause larvae throughout life, and one (No. 1) produced all its offspring as non-diapausing individuals. The remaining twelve females were of two types: nine showed a tendency to produce more diapause offspring at the end of life and the other three tended to produce more diapause larvae at the beginning of life. If the results for the seventeen females are combined (Table 3), an overall TABLE ~-SHOWING
THE EFFECTOF THE AGE OF FEMALE Nasonia vitripe-nnis UPON THE PERCENTAGEOF THE PROGENY ENTERING DIAPAUSE
(days)
No. of females alive
1 2 5 6 7 8 9 11 12 13 14 15 16 18 19 20 21 22 23 24 25 26 27 28
17 17 17 17 17 16 15 15 15 12 12 12 12 12 10 7 7 6 6 6 5 3 3 1
Age of females
Total :;a spring 32 222 231 329 312 173 298 55 173 93 85 178 128 46 167 69 91 89 89 29 20 43 25 15
Total no. adults
No. of diapause larvae
% diapause larvae
Log. % diapause larvae
27 155 178 245 236 130 214 37 89 45 32 94 68 20 88 28 25 23 29 6 0 0 1 0
5 67 53 84 76 43 84 18 84 48 53 84 60 26 79 41 66 66 60 23 20 43 24 15
IS.62 30.18 22.94 25.53 24.36 24.85 28.19 32.73 48.55 24.87 62.35 47.19 46.87 56.52 47.30 59.42 72.53 74.16 67.41 79.31 100~00 100~00 96.00 100~00
I.1937 I.4797 I.3606 I.4070 I.3867 1.3954 I.4501 I.5149 I.6861 I.3957 I.7948 I.6738 I.6709 I.7522 I.6749 I.7739 I.8605 I.8702 I.8288 1.8994 2.0000 2.0000 I.9823 2.0000
increase in the proportion of the progeny entering diapause is shown to occur as the parent females become more senile. Plotting the age of the females against the logarithm of percentage diapause (Fig. 1) results in a straight line. These results show that, for the female Nasonia investigated, there is a positive association between the age of the females at oviposition and the proportion of the resultant It is pointed out, however, that the number of females larvae entering diapause.
EFFRCT
OF AGE
OF NASONIA
V~T~P~~NIS
UPON LARVAL
315
DlAPAUSR
alive on any one day drops during the experiment from seventeen to one. This means that the reliability of the results for the older females is less than that for the younger females. . .
2.0-
.
.-
1.2-* 0
I 5
I I 15 20 IO Age of maternal generation,
25
30
days
FIG. 1. The effect of the age of the maternal generation of Nasonia oitripmnis the proportion of the progeny entering diapause.
upon
In view of the heterogeneity of the results, they were tested for significance by the following method. The life-span of each female was divided into two to give a ‘young’ half and an ‘old’ half. Knowing the overall percentage of the offspring of each female entering diapause, it was then possible to calculate the expected frequency of diapause larvae in each half life-span, assuming an even distribution between the two halves. These expected values were then compared with the observed rates of diapause and a series of x2 worked out (Table 4). These were combined by summing the x’s, dividing by ,/17 and finally squaring the figure obtained. In the case of those females which showed a drop in the proportion of progeny entering diapause as they became older, the x obtained was treated as a negative value in the summation. This procedure gave a combined xa of 95.96 (P ~O*OOl) and shows that the tendency to produce more diapause larvae in the second half of life is highly significant. DISCUSSION Several
examples of diapause being induced by factors affecting the maternal generation are now known. SIMMONDS(1946, 1948) showed that old females of Spalangia drosophilae, a pteromalid parasite of the fritfly, produced a greater proportion of diapause larvae in their offspring than young females. The results
D.
316 shown
in the present
diapause working
occurs
paper
in ?&.so&z
with N. ~~~~~n~,
oiigenesis,
or depriving
S. SAUNDERS
are evidence
that a similar
~~~~~~~~. found
them
relation
that exposing
of hosts
females
in which
between
and HORW~TZ
~CHNE~D~N
age and
(1958),
to low temperature
to oviposit,
also
also
during
induced
larval
TABLE ~-THE OBSERVEDAND EXPECTED FREQUENCIESOF DIAPAUSE LARVAE IN THE OFFSPRING OF FEMALE Nasonia VitYi@?lniS,SHOWING THAT THE GREATER PROPORTION OF DIAPAUSE LARVAE ARE PRODUCED BY A FEMALE IN THE SECOND HALF OF LIFE
Total no. in diapause
Total RO. Off-
% diapause of total
spring 1 2 3 4 5 ! 8 9 10 11 12 13 14 15 16 17
144 294 200 71 138 71 174 234 257 186 176 190 363 162 250 195 130
040 1.36 2.50 5.63 8.69 14.08 22.41 23‘93 27.63 34.95 36.34 37.89 52.62 78.39 98.00
0 4 5 4 12 10 39 56 71 65 64 72 191 127 245 195 130
100.00 100*00
No. of diapause larvae Observed Expected Xs
*
t
*
t
0 4 0 0 12 1 0 9 0 0 34 62 0 68 123 69 53
0 0 5 4 0 9 39 47 71 65 30 10 191 59 122 126 77
0 l-73 3.35 0.51 6.26 2.67 25.32 2740 32.60 35.99 37.88 43.57 85.00 79.17 125.41 69.00 53.00
0 2.27 1.6s 3.49 5.74 7.32 13.67 28.59 38.40 29.01 26.11 28-42 105.99 47.82 117.57 126.00 77.00
X
0.~ - 2.2910 3.2410 0.2821 - 3.3170 1.2830 8.4980 4.9190 7.7630 8.9440 0.9866 - 4*44-M 12.3800 2+wo O-0980 0~0000 0~0000
0.~0 5.2480 10.1492 0.0796 1 lGo20 1.6479 72.2185 24.1981 60.2760 80.~ 0.9739 19.7474 153.2100 4.1850 0.~96 o*oooo 0~0000
CX = 40.3877.
(pg =f (4Eg)” = 9.79572 = 95.96,
P 4 O*OOl.
* First haif of life-span. t Second haIf of life-span. diapause
in the
influence
on the induction
ser&-&.z
offspring.
the
Chalcidoidea,
hormone
been
shown
produced
silkworm, generation
to enter diapause
however, is most
(FUKUDA,
by the sub-oesophageal
causes the progeny hormone
in the silkworm
the stimulus
effective
(KOGURE,
to the eggs
1933) ; the stimulus
by almost a generation
1951;
HASEGAWA,
in females the egg.
which
initiates give
‘and the resultant
in this insect.
a maternal by L&&z
(ROUBAU~,
within which
of
are provided
~u~~~~~
ganglion
as embryos
(illumination)
if applied
examples
in the offspring
(CRAGG and COLE, 1952) and ~~~e~o~~~~
has also
separated
Outside
of diapause
1928).
1951)
during
It
that a
oijgenesis
In the case of the the release
of the
rise to the maternal
diapause
are therefore
EFF%CTOF AGE OF NASONM
Z?TRIPENNIS
UPON LARVAL DIAPAUSE
317
In Naso?& nitripennti, the mechanisms by which these maternal influences operate is not known. It is apparent, however, that females exposed to low temperature, or to the effects of age or host deprivation during o6genesis, must lay eggs which, because they give rise to diapausing progeny, differ in some way from normal eggs. If diapause in the larvae of NU~O&Zis under some sort of humoral control, as in several other insects (see LED, 1955), these maternal influences must affect the hormone balance of the larva by some mechanism which is transmitted through the ovary. SCHNEIDERMAN and HORW~TZpoint out that low temperature could act directly upon the ovaries during oiigenesis and result in the production of ‘diapause’ eggs, or through the mediation of another organ as in the silkworm. The same could also be said of the effect of the age of the female parent and of host deprivation. N~~~~ w~~~~~~~ occurs in several different continents and climates, and there is evidence that strain differences occur within the species. For instance, COUSIN(1933) working with N. vitripennis from France, SAUNDERS (1958) with London material, and SCHNEIDERMAN and HORWITZ (1958) working with a strain obtained from Woods Hole all record the duration of the parasitic phase of the life cycle as 14 days at 25°C. Vhr~ DER MERWE (1943), on the other hand, working in South Africa, records a slightly longer developmental period at this temperature. Furthermore, SCHNEIDERMANand HORWITZ found that chilling the female parent induced diapause in the offspring, but quote MOURSI(1946) who failed to induce diapause by this method in Californian N. vitripennnis. It is probable, therefore, that the discrepancy between the present results obtained with the London material and those of SC~NEIDERMAN and HORWITZwith the Woods Hole material is also due to differences in strain. Acknowledgements-This work was carried out in the Department of Entomology, London School of Hygiene and Tropical Medicine, whilst the author was in receipt of a Medical Research Council Scholarship. The author is grateful to Professor D. S. BERTRAM for his help during the investigation and to Dr. BRYAN CLARKE for discussing the statistical treatment of the results. REFERENCES ALTSON A. M. (1920) The life history and habits of two parasites of blowflies.
Proc. 2001.
Sot. Lond. 15, 196-243. COUSIN G. (1932) Etude experimentale de la diapause des insectes. Bull. biol. (Suppi.) 15, l-341. COUSIN G. (1933) Etude biologique d’un chalcidien: Illormortie~~aaitripennis Walk. Bull.
biol. 67, 371400. CRAGGJ. B. and COLE P. (1952) Diapause in Luciliu swicata (Mg.) Diptera. r. exp. Biol. 29, 600-604. EDWARDS R. L. (1954)
The host-finding and oviposition behaviour of ~o~on~eZla n&pipennis (Walker) (Hym. Pteromalidae), a parasite of Muscoid flies. Behaviour 7, 88-112. EVANS A. C. (1933) Comparative observations on the morphology and biology of some Hymenopterous parasites of carrion-infesting Diptera. Bull. ent. Res. 24, 385-405. FUKUDA S. (1951) Production ganglion in the silkworm.
of the diapause eggs by transplanting
Proc. imp. Acad.Japan
27, 672-677.
the subesophageal
318
D. S. SAUNDERS
HASEGAWAK. (1951) Studies in voltinism in the silkworm, Bombyx mori L., with special reference to the organs concerning determination of voltinism (A preliminary note). Proc. imp. Acad. Japan 27, 667-671. KOGURE M. (1933) The influence of light and temperature on certain characters of the silkworm, Bombyx mori. J. Dep. Agric. Kyushu Univ. 4, l-93. LEES A. D. (1955) The Physiology of Diapause in Arthropods. Cambridge University Press. MERWE J. S. VAN DER (1943) Investigations on the biology and ecology of Mormoniella vitripennis Walk. (Pteromalidae, Hym.). J. ent. Sot. S. Afr. 6, 48-64. MOUR~I A. A. (1946) The effect of temperature on development and reproduction of Mormoniella vitripennis (Walker). Bull. Sot. ent., l?gypte 30, 39-61. ROUBAUDE. (1917) Observations biologiques sur Nasonia brevicornis Ashm., Chalcidide parasite des pupes des Muscides. Detenninisme physiologique de l’instinct de ponte; adaptation a la lutte contre les glossines. Bull. sci. Fr. Belg. 1, 425-439. ROUBAUDE. (1928) Asthenobiose et hibernation obligatoire provoquee chez P. papatasii Stop. Bull. Sot. Pat. exot. 21, 436439. SAUNDERSD. S. (1958) The biology of parasites of fly puparia. Ph.D. thesis, University of London. SCHNEIDERMAN H. A. and HORWITZJ. (1958). The induction and termination of facultative diapause in the chalcid wasps Mormoniella vitripennis (Walker) and Tritneptis klugii (Ratzeburg). J. exp. Biol. 35, 520-551. SIMMONDS F. J. (1946) A factor affecting diapause in Hymenopterous parasites. Bull. ent. Res. 37, 95-97. SIMMONDS F. J. (1948) The influence of maternal physiology on the incidence of diapause. Phil. Trans. (B) 233, 385414.