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Larval diapause of maternal origin—III. The effect of host shortage on Nasonia vitripennis
r. InsectPhysiol.,1966, Vol. 12, p$. 899 to 908. PergamonPressLtd. Printedin Great Britain
LARVAL DIAPAUSE OF MATERNAL ORIGIN-III. THE EFFECT OF HOST SHORTAGE ON NASONIA VITRIPENNIS D. S. SAUNDERS Department of Zoology, University of Edinburgh (Received 7 March 1966)
Abstract-The two most important results of host shortage (small body size and host deprivation) are examined for their effect on the production of diapause larvae by the parasitic wasp, Nasonia vitripemis. Body size has a pronounced effect on longevity and fecundity but does not affect the rate of ‘switching’ to the production of diapause larvae or, therefore, the proportion of diapause larvae produced. Host deprivation, on the other hand, has little effect on longevity and fecundity but greatly increases the proportion (and number) of diapause larvae since it delays the start of oviposition without altering the age at which the ‘switch’ occurs. It is clear, therefore, that neither body size nor host deprivation affects the timing mechanism which is responsible for the ‘adding up’ of short-day cycles needed to effect the ‘switch’.
INTRODUCTION
LARVAL diapause in the parasitic wasp, Nasonia ( = Mormoniella) vitripennis, is induced by environmental factors affecting the maternal generation ( SCHNEIDERMAN and HORWITZ, 1958; SAUNDERS,1965). In conditions of long daylength and high temperature females deposit eggs which give rise to larvae which develop and pupate without arrest, but at short daylength and low temperature the females produce developing larvae for the first few days of adult life and then ‘switch’ to the production of diapause larvae. Between 5 and 11 short-day cycles are required to effect the ‘switch’ and the critical daylength is between 15 and 15i hr per day.’ Temperature modifies the effect of photoperiod by altering the rate of oviposition, whilst the number of short-day cycles needed to complete the ‘switch’ is virtually temperature-independent. Details of this induction process are given in an earlier paper in this series (SAUNDERS,1966), Apart from photoperiod and temperature the most important factor affecting the biology of a parasitic insect such as N. vitripennis is the availability of hosts, since hosts provide both food and a place in which to deposit the eggs (ROUBAUD, 1917). If blowfly pupae are readily available feeding and oviposition occur without delay. Host shortage, on the other hand, has two profound effects on the physiology of the insect. First, if a newly emerged female is deprived of hosts she starves, and the few eggs which develop from larval reserves undergo a slow cycle of resorption in the ovary (EDWARDS,1954; KING, 1963; KING and HOPKINS, 1963). 899
D. S. SAUNDERS
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Only when hosts are again available can the insect feed, mature eggs, and parasitize hosts. Second, if blowfly pupae are in short supply, several females may be induced to oviposit simultaneously on one host pupa thus giving rise to smallsized individuals in the next generation. Since both host deprivation and body size affect the number of eggs produced, and the rate at which they are deposited (VELTHUISet al., 196.5), these factors are examined in this paper for their effect on the pattern of diapause production. MATERIALS
Maintenance of N. vitripennis
AND
METHODS
and arzalysis of the progeny
The experiments described in this paper were. done with a strain of N. vitripennis (the C strain) isolated in Cambridge, England, by Dr. G. Salt in October 1961. Methods used for the culture, maintenance, and analysis of the progeny of this strain are as described by SAUNDERS (1966). All experimental females were kept at 18°C and either short daylength (LD 12 : 12) or long daylength (LD 18 : 6), and, unless purposely deprived of hosts, provided with two 3-day-old pupae of Sarcophaga barbata daily. Production and measurement of females of difwent body sixes Experimental females of different sizes were produced by allowing different numbers of wasps to oviposit simultaneously on S. barbata pupae, thus forcing them to superparasitize the available hosts. In this way, full-sized wasps were produced by allowing 1 female to oviposit in 2 hosts in 24 hr (parasite : host ratio = 1 : 2) and, similarly, two smaller size-classes were produced by allowing 1 female access to 1 host (1 : 1) and 3 females access to 1 host (3 : l), respectively. The progeny reared from these parasitized hosts were used to investigate the effect of body size on the production of diapause larvae. The size of an experimental female was recorded at death by measuring head width and the length of the forewing with a calibrated eye-piece micrometer. These two measurements were chosen because they are unaffected by age or the physiological state of the female. Examination of the ovaries In order to determine the effects of host deprivation, females of N. vitripennis were dissected in 0.9°h saline and the condition of their ovaries assessed by the method described by EDWARDS(1954). This method recognizes five categories of Half-mature eggs (category a) and threeovarian development and resorption. quarter-mature eggs (b) are recognized by the dark appearance of the yolk under transmitted light and by their staining deeply in 1% acetocarmine. They are differentiated by the ratio of oiicyte to nurse cell volume. Mature eggs (c) do not stain deeply in acetocarmine and appear white under reflected light because of their fully developed chorion. Lastly, two categories of resorbing eggs are recognized: partially resorbed eggs (d) which are flaccid and have lost their normal
LARVAL DIAPASUE OF MATERNAL ORIGIN-III
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hymenopteran shape, and eggs in a more advanced state of resorption (e) which appear as dark masses at the base of the ovariole. These types of developing and resorbing eggs are illustrated in Fig. 1.
1. The ovarioles and ovarian follicles of IV. v~tr;Pe&s, showing stages in egg development and resorption. (A) Ovariole enclosed in sheath, showing developing oocytes and nurse cells. (B) Half-mature eggs (category a). (C) Three-quartermature eggs (category b). (D) Mature eggs (category c). (E) Posterior end of an ovariole showing a mature egg and a string of follicular relics. (F) Egg in late stage of resorption (category e). (G) Eggs in early stage of resorption (category d). (fr, follicular relics; nc, nurse cells; OS, ovarian sheath.) The categories of egg development and resorption are as described by EDWARDS (1954). FIG.
In actively ovulating females long strings of follicular relics were observed at the posterior ends of the follicular tube when it was removed from the ovariole sheath. It seems that these structures, which indicate the degree of ovarian activity, were not observed by EDWARDS (1954). RESULTS
The efJect of body size Three groups of N. vitripennis females, one from each of the three size-classes, were kept at 18°C and at short daylength (LD 12 : 12). Each female within the group was placed in a 2 x + in. glass tube together with two pupae of S. barbata.
D. S.
902
SAUNDERS
These pupae were replaced daily, incubated at 25°C for 10 days, then opened to allow a record of the numbers of developing and diapausing progeny to be made. At death the size of each female was measured. Table 1 and Fig. 2 show the results of this experiment. The three size-classes were of significantly different size, whether measured by head width (F = 72.17, P< 0.001) or wing length (F = 66.71, P< 0.001). Body size had a profound effect on life-span and the number of progeny produced, thus agreeing with the results of other authors (VELTHUIS et al., 1965). Those females which were the result of the attack of three wasps on one pupa (the smallest size-class) had a mean life-span of 16.0 days and a mean fecundity of 189.8; these figures include seven females which were too small and weak to drill into the host puparium and therefore died within a few days without laying eggs. Those females in the other two size-classes produced correspondingly greater numbers of progeny and showed a greater survival rate, the largest size-class having a mean life-span of 29.1 days and a mean fecundity of 607.4. These differences in life-span (F = 129.06, P-cO-001) and fecundity (F = 41.50, P-c0.001) are highly significant. However, although body size had such a marked effect on fecundity and longevity it had no effect on the number of short-day cycles required to complete the ‘switch’ (F = 0.19) or, therefore, in the overall proportion of the larvae which entered diapause (F = 0.81). This result agrees with that obtained by SCHNEIDERMANand HORWITZ (1958) for N. vitripennis maintained in continuous darkness. TABLE ~-THE RELATIONBETWRENBODY SIZE AND LONGEVITY,FECUNDITYAND THE PRODUCTION OF DIAPAUSELARVAEBY FEMALESOF N. vitripe?ZniSAT 18°C AND SHORT DAYLENGTH (LD 12 : 12) Parental parasite : host ratio
Females (No.)
Width of head
Length of forewing
Mean adult life-span
(mm +
(XR-llA
(days ?
S.E.)
S.E.)
S.E.)
Mean age at ‘switch (days f S.E.)
Mean number offspring per female + S.E.
Offspring
’ dia:use (%)
3:l
20
0.57 + 0.013
1.50 + 0.034
16-O+ 2.25
8.1 rf: 1.03
189.8 zk 41.53
78.3
1 :l
20
0.68 + 0.008
1.76 + 0.019
24.9 of: 1.27
8.2 i 0.51
411.3 f 19.57
74.2
1:2
19
0.74 + 0.007
1.91 rt 0.015
29.1 + 1.72
7.7 + 0.56
607.4 ?C 29.94
77.3
Figure 2 shows the relation between oviposition rate and the production of diapause larvae in the three size-classes. Each female produced offspring which developed without arrest for the first few days of adult life, and then ‘switched’ to the production of diapause larvae i.n the manner described in earlier papers (SAUNDERS,1965; 1966). Therefore since body size had no effect on the age of the
LARVAL
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‘switch’ or the proportion of larvae produced in diapause, the pattern of diapause production was the same in all three size-classes. 60
40
20
5
2 ::
26
E 60 ______._.-._.._.__..c_______l
40
20
Age
of
maternal
generation,
days
FIG. 2. The effect of body size on the production of diapause larvae by females of N. nitripennis at 18°C and LD 12 : 12. (A) 20 of size-class 1 (parental parasite : host ratio 3 : 1). (B) 20 of size-class 2 (1 : 1). (C) 19 of size-class 3 (1 : 2). The solid line shows the rate of egg production, with the shaded portion representing the diapause larvae. The dotted line shows the survival rate for the females in the group.
The eflect
qf host
deprivation
Four groups of females were placed at 18°C and short daylength. One group was kept as a control and provided with two host pupae daily. The other three groups’ were deprived of hosts for 3, 5 and 7 days, respectively. Analysis of the progeny produced in this experiment gave the results shown in Table 2 and Fig. 3. The females of the control group produced a mean number of 6324 offspring in 324 days with a peak in the oviposition rate on about the tenth day. The mean age of the ‘switch’ in this group was 9.1 days and about 73 per cent of the progeny were produced in diapause. Being unable to feed, the host-deprived females starved and were unable to develop and deposit eggs until hosts were provided. Under the most severe conditions of 7 days without hosts, eight (20 per
D. S. SAUNDERS
904
cent) of the group died before a protein meal could be obtained. If these females are excluded, however, a period of host deprivation is found to have no effect on longevity (F = 2.18) or fecundity (F = 2.05) and only a marginal effect on the number of short-day cycles required to complete the ‘switch’ to diapause larvae (F = 2.73, P = 0.05). TABLE ~-THE EFFECT OF HOST DEPRIVATION ON THE PRODUCTION OF DIAPAUSELARVAEBY FEMALESOF N. Vit?7@?Z?ZiS AT 18°C
Deprived of hosts Females (days) (No.)
Short daylength (LD 12: 12)
Long daylength (LD 18: 6)
Mean adult life-span (days + S.E.)
0 3 5 7
19 20 18 31
32.0 30.0 34.3 35.6
0 7
19 10
25.9 + 1.81 28.7 + 3.08
f 1.95 * 2.22 I!I1.62 AI1.15
Mean age at ‘switch (days + S.E.)
9.1 7.7 8.2 8.5
+ 044 + O-38 +_0.41 k 0.15
22.7 (9)* 27.0 (3)*
Mean number of offspring per female f S.E.
632.8 537.5 552.6 532.0
Offspring in diapause (%)
+ 40.74 +_39.67 +_30.72 + 18.21
72.6 86.5 91.1 99-o
590.1 + 36.29 434.7 + 8144
4.6 5.9
* At long daylength only a small number of females survive long enough to show a ‘switch’ to the production of diapause larvae.
Fig. 3, however, shows that host deprivation had a marked effect on the pattern of diapause production since the absence of hosts delayed the start of oviposition without altering the age of the females at the ‘switch’. The effect of this delay was to increase the proportion (and the number) of diapause larvae produced. For instance, a 3 day period without hosts raised the proportion of diapause larvae to 86 per cent, and 5 and 7 days without hosts to 91 and 99 per cent, respectively. This difference between the proportion of diapause larvae in the four groups was tested by the method of SNEDECOR (1956) and found to be highly significant (F = 21.03, P-c O*OOl). SCHNEIDERIVIAN and HORWIT~ (1958) also showed that host deprivation raised the proportion of diapause larvae but were unaware of the overriding importance of photoperiod, or the ‘age-pattern’ whereby females ‘switch’ from developing to diapause larvae. At long daylength a period without hosts had no marked effect on the proportion of diapause larvae produced (Table 2). Although there was no significant difference seen between the number of progeny produced by the control and host-deprived groups, it was clear that starvation had a profound effect on the physiology of N. vitripennis. Examination of the ovaries (Table 3) showed that females allowed access to host pupae were
LARVAL
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60 _________________.__________~_
x
z ;P
.._____._._ ._._._ .__._.
40
20
? ._ h s b
?i .?
Age
of
maternal
generation,
days
FIG. 3. The effect of host deprivation on the production of diapause larvae by females of N. titripennis at 18°C and LD 12 : 12. (A) 19 females provided with two host pupae daily. (B) 20 females deprived of hosts for 3 days. (C) 18 females deprived of hosts for 5 days. (D) 31 females deprived of hosts for 7 days. The solid line shows the rate of egg production, with the shaded portion representing the diapause larvae. The dotted line shows the survival rate for the females in the group.
full of mature eggs and developing oiicytes. The ovaries of these females also showed long strings of follicular relics (Fig. 1E) on the posterior ends of the follicular tubes, indicating active and rapid egg production. After 3 days without hosts, however, the ovaries showed few developing oocytes, a large number of resorbing eggs, and the absence of follicular relics showed that no eggs had been passed out of the ovaries. Females deprived of hosts for 5 and 7 days showed
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906
TABLE ~-THE EFFECTOF HOSTDEPRIVATION ON OVARIAN DEVELOPMENT OF N. titri@en?ZiSAT 18°C ANDSHORTDAYLENGTH (LD 12 : 12)
Experimental conditions Newly emerged Two host pupae per day for 7 days Deprived 3 days Deprived 5 days Deprived 7 days
Females (No.)
Mean number of oscytes in categories
Mean number of progeny
(a>
(h)
(e>
(d)
(e>
12
12
8
0
0
0
-
15
38
30
15
0
0
160
15
4
0
0
5
-
10 13
8 8
5 2
9 0
28 27
22
0 7
-
(a) = hslf-mature eggs; (b) = three-quarter mature eggs; (c) = mature eggs; (d) = partially resorbed eggs; (e) = eggs in late stage of resorption. fewer developing eggs and a large number of degenerating eggs of both categories (d and e). Since it is known that the resorption of eggs provides a supplementary food source for starved females of N. vitripennis (KING and HOPKINS, 1963), it is assumed that a slow cycle of egg development and resorption occurs which enables them to survive a period of host deprivation but results in a considerable loss in eggs. This loss would account for the difference in the mean fecundity between the control and host-deprived groups as seen in Table 2, but is masked by the individual variation in the number of progeny produced. DISCUSSION The results presented in this and earlier papers in this series (SAUNDERS,1965 ; 1966) show that females of iV. vitripennis are able to ‘measure’ time. First, they can measure the duration of the light or dark period, or both light and dark periods, of the day with a considerable degree of accuracy, and thereby respond to ‘long-’ or ‘short-day’ conditions and produce developing or diapausing larvae accordingly. This type of ‘physiological clock’ is well known in other arthropods (LEES, 1960; DE WILDE, 1962) and the light receptor, if not the ‘clock’ itself, is probably located in the brain (LEES, 1964; WILLIAMS and ADKISSON, 1964). Females of iV. vitripennis are also able to ‘add up’ the number of short-day cycles received and then ‘switch’ to the production of diapause larvae. This response shows a fairly wide individual variation in the number of short-day cycles required to effect the ‘switch’ (5 to Il), but the mean value is constant, especially between replicates of the same experiment. .This phenomenon shows many of the characteristics of a physiological clock, e.g. it is virtually temperature-independent (QHJ = 1.04) (S AUNDERS,1966). However, it is clear that although this is an aspect of time measurement, it is not the clock itself but a secondary, or clock-dependent, process (B~~NNING,1960) which is associated with the expression of the clock by the production of diapause larvae. It is clearly identifiable, therefore, with the
LARVALDIAPAUSE OF MATERNAL ORIGIN-III
907
mechanism of diapause induction which probably involves the following stages: reception of the photoperiod, the production of a chemical factor by the brain or an associated structure, and the accumulation of this factor through successive shortday cycles to a threshold level in the haemolymph and developing oijcytes (SAUNDERS, 1965). The results of the present experiments also show that this aspect of time measurement is unaffected by body size or by a period of host deprivation (starvation). This observation is especially interesting since it demonstrates that the ‘clock’ continues to ‘add up’ short-day cycles during a period of great physiological stress when the females are being starved and are unable to produce eggs. It also shows that the mechanism of diapause induction is not associated with ovarian function alone. It is almost certainly initiated in the brain or an associated structure, and the ovaries merely express the effect of short- or long-day cycles by the production of eggs which give rise to diapause or developing progeny. The ecological importance of these results is obvious. A period of host deprivation, which is most likely to occur when the newly emerged females disperse to find new hosts, increases the proportion (and number) of diapause larvae produced. At long daylength this effect is not seen. Its effect becomes most important in the autumn when the daylength reaches the critical value (15 to 159 hr per day), and available host pupae become scarce due to a recession in the breeding blowfly population. In addition, many of the blowfly hosts of N. vitripennis enter hibernation themselves in the autumn, either as larvae (Cdiphora vi&a, C. vomitoris), prepupae (Lucilia sericata) or adults (C. vicina, Phormia terrae-novae, P. regina) (NORRIS, 1965), thus reducing the number of pupae available as hosts. It is clear therefore that short daylength and host shortage act synergistically in the autumn to increase the number of diapause larvae produced. Acknowledgements-The author would like to thank Miss M. H. MCINTYRJZ for technical assistance. This work was supported by a grant from the Science Research Council.
REFERENCES BINNING E. (1960) Opening address: Biological clocks. Cold Spring Harb. Symp. quunt. Biol. 25, l-9. EDWARDSR. L. (1954) The effect of diet on egg maturation and resorption in Mormoniella v&pen&s (Hymenoptera, Pteromalidae). Quart.J. micr. Sti. 95, 459-468. KING P. E. (1963) The rate of egg resorption in Nasoniu vitripennis (Walker) (Hymenoptera: Pteromalidae) deprived of hosts. Proc. R. ent. Sot. Lond. (A) 38, 98-100. KING P. E. and HOPKINS C. R. (1963) Length of life of the sexes in Nusoniu vitripennis (Walker) (Hymenoptera: Pteromalidae) under conditions of starvation. J. exp. BioE. 40, 751-761. LEES A. D. (1960) Some aspects of animal photoperiodism. Cold Spring Harb. Symp. quad Biol. 25, 261-268. LEES A. D. (1964) The location of the photoperiodic receptors in the aphid Megozrra vi&e Buckton. J. exp. Biol. 41, 119-133. NORRISK. R. (1965) The bionomics of blow flies. A. Rev. Ent. 10,47-68. 56
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ROUBAUDE. (1917) Observations biologiques sur Nusoniu brevicornis Ashm., ChaIcidide parasite des pupes des Muscides. Determinisme physiologique de l’instinct de ponte; adaptation a la lutte contre les gIossines. Bull. scient. Fr. Belg. 1, 425-439. SAUNDERS D. S. (1965) Larval diapause of maternal origin: Induction of diapause in Nasonzir vitripennis (Walker) (Hymenoptera: Pteromalidae). r. exp. Biol. 42,495-508. SAUNDERY, D. S. (1966) Larval diapause of maternal origin-II. The effect of photoperiod and temperature on iVasonia vitripennis. J. Insect Physiol. 12, 569-581. SCHNEIDERMAN H. A. and HORWITZ J. (1958) The induction and termination of facultative diapause in the chalcid wasps Mormoniella vitripennis (Walker) and Tritneptis klugii (Ratzeburg). 2. exp. Biol. 35, 520-551. SNEDECOR G. W. (1956) Statistical Methods. 5th ed. Iowa State University Press, Ames. VELTHUISH. H. W., VELTHUIS-KLUPPELL F. M., and BOSSINKG. A. H. (1965) Some aspects of the biology and population dynamics of Nasoniu vitripennis (WaIker) (Hymenoptera: Pteromalidae). Entomologiu exp. appl. 8, 205-227. DE WILDE J. (1962) Photoperiodism in insects and mites. A. Rev. Ent. 7, l-26. WILLIAMS C. M. and ADKISSONP. L. (1964) Physiology of insect diapaus_XIV. An endocrine mechanism for the photoperiodic control of pupal diapause in the oak silkworm, Antheraea pernyi. Biol. Bull., Woods Hole 127, 511-525.
LARVAL DIAPAUSE OF MATERNAL ORIGIN-III. THE EFFECT OF HOST SHORTAGE ON NASONIA VITRIPENNIS D. S. SAUNDERS Department of Zoology, University of Edinburgh (Received 7 March 1966)
Abstract-The two most important results of host shortage (small body size and host deprivation) are examined for their effect on the production of diapause larvae by the parasitic wasp, Nasonia vitripemis. Body size has a pronounced effect on longevity and fecundity but does not affect the rate of ‘switching’ to the production of diapause larvae or, therefore, the proportion of diapause larvae produced. Host deprivation, on the other hand, has little effect on longevity and fecundity but greatly increases the proportion (and number) of diapause larvae since it delays the start of oviposition without altering the age at which the ‘switch’ occurs. It is clear, therefore, that neither body size nor host deprivation affects the timing mechanism which is responsible for the ‘adding up’ of short-day cycles needed to effect the ‘switch’.
INTRODUCTION
LARVAL diapause in the parasitic wasp, Nasonia ( = Mormoniella) vitripennis, is induced by environmental factors affecting the maternal generation ( SCHNEIDERMAN and HORWITZ, 1958; SAUNDERS,1965). In conditions of long daylength and high temperature females deposit eggs which give rise to larvae which develop and pupate without arrest, but at short daylength and low temperature the females produce developing larvae for the first few days of adult life and then ‘switch’ to the production of diapause larvae. Between 5 and 11 short-day cycles are required to effect the ‘switch’ and the critical daylength is between 15 and 15i hr per day.’ Temperature modifies the effect of photoperiod by altering the rate of oviposition, whilst the number of short-day cycles needed to complete the ‘switch’ is virtually temperature-independent. Details of this induction process are given in an earlier paper in this series (SAUNDERS,1966), Apart from photoperiod and temperature the most important factor affecting the biology of a parasitic insect such as N. vitripennis is the availability of hosts, since hosts provide both food and a place in which to deposit the eggs (ROUBAUD, 1917). If blowfly pupae are readily available feeding and oviposition occur without delay. Host shortage, on the other hand, has two profound effects on the physiology of the insect. First, if a newly emerged female is deprived of hosts she starves, and the few eggs which develop from larval reserves undergo a slow cycle of resorption in the ovary (EDWARDS,1954; KING, 1963; KING and HOPKINS, 1963). 899
D. S. SAUNDERS
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Only when hosts are again available can the insect feed, mature eggs, and parasitize hosts. Second, if blowfly pupae are in short supply, several females may be induced to oviposit simultaneously on one host pupa thus giving rise to smallsized individuals in the next generation. Since both host deprivation and body size affect the number of eggs produced, and the rate at which they are deposited (VELTHUISet al., 196.5), these factors are examined in this paper for their effect on the pattern of diapause production. MATERIALS
Maintenance of N. vitripennis
AND
METHODS
and arzalysis of the progeny
The experiments described in this paper were. done with a strain of N. vitripennis (the C strain) isolated in Cambridge, England, by Dr. G. Salt in October 1961. Methods used for the culture, maintenance, and analysis of the progeny of this strain are as described by SAUNDERS (1966). All experimental females were kept at 18°C and either short daylength (LD 12 : 12) or long daylength (LD 18 : 6), and, unless purposely deprived of hosts, provided with two 3-day-old pupae of Sarcophaga barbata daily. Production and measurement of females of difwent body sixes Experimental females of different sizes were produced by allowing different numbers of wasps to oviposit simultaneously on S. barbata pupae, thus forcing them to superparasitize the available hosts. In this way, full-sized wasps were produced by allowing 1 female to oviposit in 2 hosts in 24 hr (parasite : host ratio = 1 : 2) and, similarly, two smaller size-classes were produced by allowing 1 female access to 1 host (1 : 1) and 3 females access to 1 host (3 : l), respectively. The progeny reared from these parasitized hosts were used to investigate the effect of body size on the production of diapause larvae. The size of an experimental female was recorded at death by measuring head width and the length of the forewing with a calibrated eye-piece micrometer. These two measurements were chosen because they are unaffected by age or the physiological state of the female. Examination of the ovaries In order to determine the effects of host deprivation, females of N. vitripennis were dissected in 0.9°h saline and the condition of their ovaries assessed by the method described by EDWARDS(1954). This method recognizes five categories of Half-mature eggs (category a) and threeovarian development and resorption. quarter-mature eggs (b) are recognized by the dark appearance of the yolk under transmitted light and by their staining deeply in 1% acetocarmine. They are differentiated by the ratio of oiicyte to nurse cell volume. Mature eggs (c) do not stain deeply in acetocarmine and appear white under reflected light because of their fully developed chorion. Lastly, two categories of resorbing eggs are recognized: partially resorbed eggs (d) which are flaccid and have lost their normal
LARVAL DIAPASUE OF MATERNAL ORIGIN-III
901
hymenopteran shape, and eggs in a more advanced state of resorption (e) which appear as dark masses at the base of the ovariole. These types of developing and resorbing eggs are illustrated in Fig. 1.
1. The ovarioles and ovarian follicles of IV. v~tr;Pe&s, showing stages in egg development and resorption. (A) Ovariole enclosed in sheath, showing developing oocytes and nurse cells. (B) Half-mature eggs (category a). (C) Three-quartermature eggs (category b). (D) Mature eggs (category c). (E) Posterior end of an ovariole showing a mature egg and a string of follicular relics. (F) Egg in late stage of resorption (category e). (G) Eggs in early stage of resorption (category d). (fr, follicular relics; nc, nurse cells; OS, ovarian sheath.) The categories of egg development and resorption are as described by EDWARDS (1954). FIG.
In actively ovulating females long strings of follicular relics were observed at the posterior ends of the follicular tube when it was removed from the ovariole sheath. It seems that these structures, which indicate the degree of ovarian activity, were not observed by EDWARDS (1954). RESULTS
The efJect of body size Three groups of N. vitripennis females, one from each of the three size-classes, were kept at 18°C and at short daylength (LD 12 : 12). Each female within the group was placed in a 2 x + in. glass tube together with two pupae of S. barbata.
D. S.
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SAUNDERS
These pupae were replaced daily, incubated at 25°C for 10 days, then opened to allow a record of the numbers of developing and diapausing progeny to be made. At death the size of each female was measured. Table 1 and Fig. 2 show the results of this experiment. The three size-classes were of significantly different size, whether measured by head width (F = 72.17, P< 0.001) or wing length (F = 66.71, P< 0.001). Body size had a profound effect on life-span and the number of progeny produced, thus agreeing with the results of other authors (VELTHUIS et al., 1965). Those females which were the result of the attack of three wasps on one pupa (the smallest size-class) had a mean life-span of 16.0 days and a mean fecundity of 189.8; these figures include seven females which were too small and weak to drill into the host puparium and therefore died within a few days without laying eggs. Those females in the other two size-classes produced correspondingly greater numbers of progeny and showed a greater survival rate, the largest size-class having a mean life-span of 29.1 days and a mean fecundity of 607.4. These differences in life-span (F = 129.06, P-cO-001) and fecundity (F = 41.50, P-c0.001) are highly significant. However, although body size had such a marked effect on fecundity and longevity it had no effect on the number of short-day cycles required to complete the ‘switch’ (F = 0.19) or, therefore, in the overall proportion of the larvae which entered diapause (F = 0.81). This result agrees with that obtained by SCHNEIDERMANand HORWITZ (1958) for N. vitripennis maintained in continuous darkness. TABLE ~-THE RELATIONBETWRENBODY SIZE AND LONGEVITY,FECUNDITYAND THE PRODUCTION OF DIAPAUSELARVAEBY FEMALESOF N. vitripe?ZniSAT 18°C AND SHORT DAYLENGTH (LD 12 : 12) Parental parasite : host ratio
Females (No.)
Width of head
Length of forewing
Mean adult life-span
(mm +
(XR-llA
(days ?
S.E.)
S.E.)
S.E.)
Mean age at ‘switch (days f S.E.)
Mean number offspring per female + S.E.
Offspring
’ dia:use (%)
3:l
20
0.57 + 0.013
1.50 + 0.034
16-O+ 2.25
8.1 rf: 1.03
189.8 zk 41.53
78.3
1 :l
20
0.68 + 0.008
1.76 + 0.019
24.9 of: 1.27
8.2 i 0.51
411.3 f 19.57
74.2
1:2
19
0.74 + 0.007
1.91 rt 0.015
29.1 + 1.72
7.7 + 0.56
607.4 ?C 29.94
77.3
Figure 2 shows the relation between oviposition rate and the production of diapause larvae in the three size-classes. Each female produced offspring which developed without arrest for the first few days of adult life, and then ‘switched’ to the production of diapause larvae i.n the manner described in earlier papers (SAUNDERS,1965; 1966). Therefore since body size had no effect on the age of the
LARVAL
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‘switch’ or the proportion of larvae produced in diapause, the pattern of diapause production was the same in all three size-classes. 60
40
20
5
2 ::
26
E 60 ______._.-._.._.__..c_______l
40
20
Age
of
maternal
generation,
days
FIG. 2. The effect of body size on the production of diapause larvae by females of N. nitripennis at 18°C and LD 12 : 12. (A) 20 of size-class 1 (parental parasite : host ratio 3 : 1). (B) 20 of size-class 2 (1 : 1). (C) 19 of size-class 3 (1 : 2). The solid line shows the rate of egg production, with the shaded portion representing the diapause larvae. The dotted line shows the survival rate for the females in the group.
The eflect
qf host
deprivation
Four groups of females were placed at 18°C and short daylength. One group was kept as a control and provided with two host pupae daily. The other three groups’ were deprived of hosts for 3, 5 and 7 days, respectively. Analysis of the progeny produced in this experiment gave the results shown in Table 2 and Fig. 3. The females of the control group produced a mean number of 6324 offspring in 324 days with a peak in the oviposition rate on about the tenth day. The mean age of the ‘switch’ in this group was 9.1 days and about 73 per cent of the progeny were produced in diapause. Being unable to feed, the host-deprived females starved and were unable to develop and deposit eggs until hosts were provided. Under the most severe conditions of 7 days without hosts, eight (20 per
D. S. SAUNDERS
904
cent) of the group died before a protein meal could be obtained. If these females are excluded, however, a period of host deprivation is found to have no effect on longevity (F = 2.18) or fecundity (F = 2.05) and only a marginal effect on the number of short-day cycles required to complete the ‘switch’ to diapause larvae (F = 2.73, P = 0.05). TABLE ~-THE EFFECT OF HOST DEPRIVATION ON THE PRODUCTION OF DIAPAUSELARVAEBY FEMALESOF N. Vit?7@?Z?ZiS AT 18°C
Deprived of hosts Females (days) (No.)
Short daylength (LD 12: 12)
Long daylength (LD 18: 6)
Mean adult life-span (days + S.E.)
0 3 5 7
19 20 18 31
32.0 30.0 34.3 35.6
0 7
19 10
25.9 + 1.81 28.7 + 3.08
f 1.95 * 2.22 I!I1.62 AI1.15
Mean age at ‘switch (days + S.E.)
9.1 7.7 8.2 8.5
+ 044 + O-38 +_0.41 k 0.15
22.7 (9)* 27.0 (3)*
Mean number of offspring per female f S.E.
632.8 537.5 552.6 532.0
Offspring in diapause (%)
+ 40.74 +_39.67 +_30.72 + 18.21
72.6 86.5 91.1 99-o
590.1 + 36.29 434.7 + 8144
4.6 5.9
* At long daylength only a small number of females survive long enough to show a ‘switch’ to the production of diapause larvae.
Fig. 3, however, shows that host deprivation had a marked effect on the pattern of diapause production since the absence of hosts delayed the start of oviposition without altering the age of the females at the ‘switch’. The effect of this delay was to increase the proportion (and the number) of diapause larvae produced. For instance, a 3 day period without hosts raised the proportion of diapause larvae to 86 per cent, and 5 and 7 days without hosts to 91 and 99 per cent, respectively. This difference between the proportion of diapause larvae in the four groups was tested by the method of SNEDECOR (1956) and found to be highly significant (F = 21.03, P-c O*OOl). SCHNEIDERIVIAN and HORWIT~ (1958) also showed that host deprivation raised the proportion of diapause larvae but were unaware of the overriding importance of photoperiod, or the ‘age-pattern’ whereby females ‘switch’ from developing to diapause larvae. At long daylength a period without hosts had no marked effect on the proportion of diapause larvae produced (Table 2). Although there was no significant difference seen between the number of progeny produced by the control and host-deprived groups, it was clear that starvation had a profound effect on the physiology of N. vitripennis. Examination of the ovaries (Table 3) showed that females allowed access to host pupae were
LARVAL
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OF MATRRNAL
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60 _________________.__________~_
x
z ;P
.._____._._ ._._._ .__._.
40
20
? ._ h s b
?i .?
Age
of
maternal
generation,
days
FIG. 3. The effect of host deprivation on the production of diapause larvae by females of N. titripennis at 18°C and LD 12 : 12. (A) 19 females provided with two host pupae daily. (B) 20 females deprived of hosts for 3 days. (C) 18 females deprived of hosts for 5 days. (D) 31 females deprived of hosts for 7 days. The solid line shows the rate of egg production, with the shaded portion representing the diapause larvae. The dotted line shows the survival rate for the females in the group.
full of mature eggs and developing oiicytes. The ovaries of these females also showed long strings of follicular relics (Fig. 1E) on the posterior ends of the follicular tubes, indicating active and rapid egg production. After 3 days without hosts, however, the ovaries showed few developing oocytes, a large number of resorbing eggs, and the absence of follicular relics showed that no eggs had been passed out of the ovaries. Females deprived of hosts for 5 and 7 days showed
D. S. SAUNDERS
906
TABLE ~-THE EFFECTOF HOSTDEPRIVATION ON OVARIAN DEVELOPMENT OF N. titri@en?ZiSAT 18°C ANDSHORTDAYLENGTH (LD 12 : 12)
Experimental conditions Newly emerged Two host pupae per day for 7 days Deprived 3 days Deprived 5 days Deprived 7 days
Females (No.)
Mean number of oscytes in categories
Mean number of progeny
(a>
(h)
(e>
(d)
(e>
12
12
8
0
0
0
-
15
38
30
15
0
0
160
15
4
0
0
5
-
10 13
8 8
5 2
9 0
28 27
22
0 7
-
(a) = hslf-mature eggs; (b) = three-quarter mature eggs; (c) = mature eggs; (d) = partially resorbed eggs; (e) = eggs in late stage of resorption. fewer developing eggs and a large number of degenerating eggs of both categories (d and e). Since it is known that the resorption of eggs provides a supplementary food source for starved females of N. vitripennis (KING and HOPKINS, 1963), it is assumed that a slow cycle of egg development and resorption occurs which enables them to survive a period of host deprivation but results in a considerable loss in eggs. This loss would account for the difference in the mean fecundity between the control and host-deprived groups as seen in Table 2, but is masked by the individual variation in the number of progeny produced. DISCUSSION The results presented in this and earlier papers in this series (SAUNDERS,1965 ; 1966) show that females of iV. vitripennis are able to ‘measure’ time. First, they can measure the duration of the light or dark period, or both light and dark periods, of the day with a considerable degree of accuracy, and thereby respond to ‘long-’ or ‘short-day’ conditions and produce developing or diapausing larvae accordingly. This type of ‘physiological clock’ is well known in other arthropods (LEES, 1960; DE WILDE, 1962) and the light receptor, if not the ‘clock’ itself, is probably located in the brain (LEES, 1964; WILLIAMS and ADKISSON, 1964). Females of iV. vitripennis are also able to ‘add up’ the number of short-day cycles received and then ‘switch’ to the production of diapause larvae. This response shows a fairly wide individual variation in the number of short-day cycles required to effect the ‘switch’ (5 to Il), but the mean value is constant, especially between replicates of the same experiment. .This phenomenon shows many of the characteristics of a physiological clock, e.g. it is virtually temperature-independent (QHJ = 1.04) (S AUNDERS,1966). However, it is clear that although this is an aspect of time measurement, it is not the clock itself but a secondary, or clock-dependent, process (B~~NNING,1960) which is associated with the expression of the clock by the production of diapause larvae. It is clearly identifiable, therefore, with the
LARVALDIAPAUSE OF MATERNAL ORIGIN-III
907
mechanism of diapause induction which probably involves the following stages: reception of the photoperiod, the production of a chemical factor by the brain or an associated structure, and the accumulation of this factor through successive shortday cycles to a threshold level in the haemolymph and developing oijcytes (SAUNDERS, 1965). The results of the present experiments also show that this aspect of time measurement is unaffected by body size or by a period of host deprivation (starvation). This observation is especially interesting since it demonstrates that the ‘clock’ continues to ‘add up’ short-day cycles during a period of great physiological stress when the females are being starved and are unable to produce eggs. It also shows that the mechanism of diapause induction is not associated with ovarian function alone. It is almost certainly initiated in the brain or an associated structure, and the ovaries merely express the effect of short- or long-day cycles by the production of eggs which give rise to diapause or developing progeny. The ecological importance of these results is obvious. A period of host deprivation, which is most likely to occur when the newly emerged females disperse to find new hosts, increases the proportion (and number) of diapause larvae produced. At long daylength this effect is not seen. Its effect becomes most important in the autumn when the daylength reaches the critical value (15 to 159 hr per day), and available host pupae become scarce due to a recession in the breeding blowfly population. In addition, many of the blowfly hosts of N. vitripennis enter hibernation themselves in the autumn, either as larvae (Cdiphora vi&a, C. vomitoris), prepupae (Lucilia sericata) or adults (C. vicina, Phormia terrae-novae, P. regina) (NORRIS, 1965), thus reducing the number of pupae available as hosts. It is clear therefore that short daylength and host shortage act synergistically in the autumn to increase the number of diapause larvae produced. Acknowledgements-The author would like to thank Miss M. H. MCINTYRJZ for technical assistance. This work was supported by a grant from the Science Research Council.
REFERENCES BINNING E. (1960) Opening address: Biological clocks. Cold Spring Harb. Symp. quunt. Biol. 25, l-9. EDWARDSR. L. (1954) The effect of diet on egg maturation and resorption in Mormoniella v&pen&s (Hymenoptera, Pteromalidae). Quart.J. micr. Sti. 95, 459-468. KING P. E. (1963) The rate of egg resorption in Nasoniu vitripennis (Walker) (Hymenoptera: Pteromalidae) deprived of hosts. Proc. R. ent. Sot. Lond. (A) 38, 98-100. KING P. E. and HOPKINS C. R. (1963) Length of life of the sexes in Nusoniu vitripennis (Walker) (Hymenoptera: Pteromalidae) under conditions of starvation. J. exp. BioE. 40, 751-761. LEES A. D. (1960) Some aspects of animal photoperiodism. Cold Spring Harb. Symp. quad Biol. 25, 261-268. LEES A. D. (1964) The location of the photoperiodic receptors in the aphid Megozrra vi&e Buckton. J. exp. Biol. 41, 119-133. NORRISK. R. (1965) The bionomics of blow flies. A. Rev. Ent. 10,47-68. 56
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ROUBAUDE. (1917) Observations biologiques sur Nusoniu brevicornis Ashm., ChaIcidide parasite des pupes des Muscides. Determinisme physiologique de l’instinct de ponte; adaptation a la lutte contre les gIossines. Bull. scient. Fr. Belg. 1, 425-439. SAUNDERS D. S. (1965) Larval diapause of maternal origin: Induction of diapause in Nasonzir vitripennis (Walker) (Hymenoptera: Pteromalidae). r. exp. Biol. 42,495-508. SAUNDERY, D. S. (1966) Larval diapause of maternal origin-II. The effect of photoperiod and temperature on iVasonia vitripennis. J. Insect Physiol. 12, 569-581. SCHNEIDERMAN H. A. and HORWITZ J. (1958) The induction and termination of facultative diapause in the chalcid wasps Mormoniella vitripennis (Walker) and Tritneptis klugii (Ratzeburg). 2. exp. Biol. 35, 520-551. SNEDECOR G. W. (1956) Statistical Methods. 5th ed. Iowa State University Press, Ames. VELTHUISH. H. W., VELTHUIS-KLUPPELL F. M., and BOSSINKG. A. H. (1965) Some aspects of the biology and population dynamics of Nasoniu vitripennis (WaIker) (Hymenoptera: Pteromalidae). Entomologiu exp. appl. 8, 205-227. DE WILDE J. (1962) Photoperiodism in insects and mites. A. Rev. Ent. 7, l-26. WILLIAMS C. M. and ADKISSONP. L. (1964) Physiology of insect diapaus_XIV. An endocrine mechanism for the photoperiodic control of pupal diapause in the oak silkworm, Antheraea pernyi. Biol. Bull., Woods Hole 127, 511-525.