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Attributes of oviposition substrates affect fecundity in Rhodnius prolixus
Pergamon PII: SOO22-1910(96)00043-l
1996 J. Insect Physiol. Vol. 42, No. 9, pp. 837-841, Copyright 0 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910196 $15.00 + 0.00
Attributes of Oviposition Substrates Affect Fecundity in Rhodnius prolixus PABLO
E. SCHILMAN,*t
Received
8 November
JOSUh
A. NtJfdEZ,*
1995; revised and accepted
CLAUDIO
26 February
R. LAZZARI*
1996
The effect of the oviposition substrate on the rate of egg laying was investigated in the haematophagous bug Rhodnius prolixus. Fresh or old hen feathers or corrugated cardboard were offered to ovipositing females. The insects showed significant differences in the number of eggs laid depending on the substrate. Egg laying was maximal on the fresh feathers and minimal on the cardboard. The variations cannot be explained by differences in the amount of blood ingested nor by differences in the mating frequency between treatments. The amount of blood necessary per laid egg exhibited an inverse relationship with the absolute fecundity. The dynamics of,mating and egg-laying for groups having two different substrates (corrugated cardboard and hen feather) and different nutritional states (previously starved vs previously fed) was established throughout one month. After a single meal, previously starved insects having feathers as a substrate laid their eggs and reached an oviposition peak sooner (6 days) than previously starved insects on the cardboard substrate. For both nutritional states the lack of feathers decreased the cumulative number of eggs laid by one third. Results are discussed in relation to the mechanisms of ovarian regulation known at present. Copyright 0 1996 Elsevier Science Ltd R. prolixus
Mating
Fecundity
Oviposition
substrates
INTRODUCTION
The selection of suitable oviposition sites by female insects is of great importance to ensure that the eggs would be adequately protected from the environment and that food will be available for the larvae when they hatch. Triatominae exhibit two different strategies of oviposition: (1) species associated with birds attach their eggs to the feathers of their host; (2) other species lay their eggs in crevices (Lent and Wygodzinsky, 1979). Rhodnius prolixus, an important vector of Chagas’ disease in the north of South America and a classical model in insect physiology, belongs to the first group. In nature, this species inhabits birds nests, but it is also found in human housings due to passive transport or active displacement (Lent and Wygodzinsky, 1979). Feeding and mating are involved in the modulation of ovarian function in R. prolixus. As in most insects, the development of oocytes in this bug is under hormonal
*Laboratorio de Fisiologia de Insectos, Departamento de Cs. BioKgicas, Facultad de Cs. Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria (1428), Buenos Aires, Argentina. tTo whom all correspondence should be addressed. 837
Ovarian function
Chagas’
disease
control and involves cerebral neurosecretory cells and the corpora allata. In R. prolixus a batch of eggs is laid following each blood meal, and the number of eggs depends on the size of the meal ingested (Buxton, 1930). Feeding promotes the secretion of juvenile hormone, which regulates vitellogenin production by the fat body (Coles, 1965), previtellogenic development of the oocytes and vitellogenesis. In the latter case, the juvenile hormone induces the separation of the follicle cells from each other, so that haemolymph proteins have free access to the oocytes (Abu-Hakima and Davey, 1977). Mating, on the other hand, provokes the release of blood-borne substances from the spermathecae of the mated female, which stimulate the neurosecretory cells of the pars intercerebralis of the brain. As a response, these release a myotropic factor that increases the motility of the oviduct muscles (Kriger and Davey, 1982). The presence of mature eggs in the ovary causes peripheral neurosecretory cells to secrete an antigonadotropic factor, which blocks vitellogenesis in younger oocytes (Davey and Kuster, 1981). In this report, we analyse the effect of an external input other than mating and feeding, i.e. the availability of different substrates for oviposition on the ovarian function of R. prolixus. In a first series of experiments, different
PABLO E. SCHILMAN et al.
838
substrates were offered to females of R. prolixus and changes in the fecundity (egg laying) and blood-egg conversion were analysed. In a second series of experiments, the dynamics of copulation and oviposition under the different treatments was studied. MATERIALS AND METHODS Adults of R. prolixus, 2-3 months old, reared in the laboratory at 28°C and constant dark regime, were used throughout. The insects were fed weekly with heparinized bovine blood using an artificial feeder (Nuiiez and Lazzari, 1990) as nymphs and adults. Adults were maintained in plastic recipients of 500 ml capacity in groups of about 15 females and 15 males. A piece of corrugated cardboard and hen feathers were present inside each recipient. At the beginning of the experiments, females were mated and had been through about 8-l 2 oviposition cycles. Insects were distributed in 15 equal sized compartments (5.5 x 5.5 x 4.5 cm) of a transparent acrylic box of 22.5 x 17.5 x 4.5 cm. The acrylic cover was perforated to allow air circulation. The floor was coated with filter paper. The acrylic box was kept at 28°C under a constant dark regime. First experimental series In this series, the fecundity (number of eggs laid) of females in the presence of different oviposition substrates was analysed. Females were marked with acrylic paint for identification and individually weighed to the nearest 0.1 mg. They were placed in a communal jar for feeding for two consecutive days (afternoon and next morning) to ensure feeding of a high proportion of insects. Three hours after the second meal, they were weighed again and the amount of blood ingested by each insect‘calculated. Male insects were fed at the same time as the females in a different communal jar. Thereafter, groups of one female and two males were put together in each compartment with a given substrate for oviposition (day 0). Three different substrates were offered: (1) a piece of corrugated cardboard and a feather removed from a hen within 24h before the beginning of the experiment (henceforth: fresh feather); (2) a piece of corrugated cardboard and a feather removed from a hen at least one month before the beginning of the experiment (henceforth: old feather); and (3) two pieces of corrugated cardboard. The feathers were wrapped with aluminium foil and kept until the experiment began. Each treatment lasted 1 week and was repeated in three successive weeks changing the oviposition substrate, so that every female was exposed to all different substrates avoiding individual variation. The chambers were washed between treatments. Insects were fed once a week as described and the eggs laid during the time they were in the communal feeding jar were not considered in the calculations. Number of eggs laid per week as related to substrates
for oviposition (fresh feather, old feather and cardboard) was statistically analysed by means of nonparametric randomized block analysis of variance (Friedman test). Multiple comparisons between groups were performed by a posteriori Dunn’s test (Zar, 1984). Two replications of this experimental series were made (n = 15 females and 30 males) involving a total number of 30 females and 60 males. Females that did not feed were excluded from the analysis to avoid any increase in variability by a factor (feeding) that must be under control. As a consequence, the initial number of females used (30) was reduced to 12. Second experimental series The dynamics of egg laying and mating was analysed for two different substrates (cardboard and fresh feather) and for insects differing in their nutritional state. Two groups of 15 mated females and 30 males were used, each differing in its nutritional history. One group was fed weekly since the imaginal ecdysis and displayed a continuous oviposition behaviour for about 8-l 2 weeks before the experiment began. The other group was not fed for one month and showed sporadic oviposition previous to the beginning of the experiment. Bugs belonging to the latter group had about 18 days of true starvation. This is due to the fact that R. prolixus takes a large blood meal which is stored in the crop and animals can only be considered as ‘starved’ when these reserves are exhausted. In a mated female, this occurs in the 12th day after feeding (Pratt and Davey, 1972b). The two nutritional states used in this experiment are thus equivalent to 0 and 18 days of starvation. In this series, we analysed the incidence of the characteristics of the substrate and nutritional state in the dynamics of egg laying and mating. Insects were fed once at the beginning (day 0), as described in the first series, and not fed until the end of the experiment (day 30). Two replications of each assay were made. In the group fed weekly prior to the beginning of the experiment, a total of 30 females (15 cardboard + 15 feather) and 60 males were used. At the end of the experimental period (1 month), 28 females of this group were taken into account, because two cardboard insects died. Different insects were used in each replication. In the ‘starved’ group, 15 females and 30 males were observed throughout one month. Thereafter the insects were fed, groups interchanged (cardboard and feather) and the insects tested for another 30 days. During the course of the experiment, 1 feather and 2 cardboard insects died. The number of eggs laid by each female and the spermatophores found in each compartment were recorded every 3 days. Data were statistically analysed by means of a twofactor analysis of variance with equal replication.
SUBSTRATE-DEPENDENT
FECUNDITY
IN R. PROLIXUS
839
RESULTS First experimental
series
The number of eggs laid was significantly different depending on the oviposition substrate available (Table 1). The number of eggs laid by the cardboard group differed significantly from that of the fresh feather group and old feather group. No significant difference was found between old and fresh feather groups. The differences in the amount of eggs laid were independent of the amount of blood ingested (Table 1) and were therefore due to the nature of the substrate offered. Differences in the conversion of blood ingested into eggs laid were evinced between groups (Table 1). As the ratio was calculated from total amounts of laid eggs and blood ingested by the 12 females, no standard errors were obtained and, therefore, no statistical analysis could be applied. The reason for this procedure was the fact that although all the females ate, not all of them oviposited. Second experimental
series
In the long-term assay, the number of spermatophores and the number of eggs laid were analysed over a month (Fig. 1). Concerning egg laying, ANOVA analysis revealed significant effects of the treatments (cardboard vs feather, F = 7.284, df = 1, P = O.Ol), but not of the previous nutritional status (weekly fed vs starved, F = 2.418, df = 1, P = 0.127), nor of the interaction between factors (treatment and nutritional status, F = 0.352, df = 1, P = 0.556). Differences within groups were revealed as a function of time (days of experiment, F = 4.028, df = 8, P
TABLE
1. Relationship
between
blood
Treatment
Females
Cardboard Old feather Fresh feather Friedman test
12 12 12
(n)
ingestion
0
3
6
9
12
15
Days after feeding FIGURE 1. The number of eggs laid (bar plot; x + SE) and the number of spermatophores (line plot; R ? SE) for both substrate treatments (cardboard and feather) as related to the time after feeding. The sampling interval was 3 days. (a) animals were fed weekly and had a continuous oviposition activity previous to the beginning of the experiment; (b) animals were fasted for 1 month (18 days of starvation) and had a sporadic or no oviposition before the experiment began.
Statistical analysis (ANOVA) revealed differences in the dynamics according to both treatments (F = 3.920, df = 8, P
and egg production by R. oviposition
prolixus females and the availability
of different
Meal size (mg)
Eggs laid
mg blood/egg
37.78k4.19 49.15f7.74 47.92H4.21 ,+4.979, df=2, ns
lf0.37 10.67ti.10 17.25+3.19 ,$=11.787,
31.76 4.61 2.18
substrates
for
df=2, p
Significant differences in the number of eggs laid but not in the meal size were found among treatments “Dunn’s test a posteriori (df = 3) fresh feather vs cardboard, Q = 3.866, P
Q = 2.762, P cO.05;
old vs
PABLO E. SCHILMAN et al.
840
Although the fertility of the eggs was not quantified, most of them hatched in both groups and treatments, indicating that a successful sperm transference took place. The analysis of the dynamics of accumulated eggs [Fig. 2(a, b)] reveals that cardboard females laid about one-third less eggs than the feather females. A significant difference was found between cardboard and feather in both nutritional states [Fig. 2(a): t = 2.197, df = 26, P CO.05 and Fig. 2(b): t = 2.359, df = 25, P cO.051. No significant difference was found between cardboard and feather treatments in the amount of blood ingested in both nutritional states (unpaired t-test, data not shown). DISCUSSION
Up to now, the mechanisms controlling egg production have been analysed in terms of internal regulation paths (i.e. nervous, endocrine), modulated by mating and feeding. Our results show that external inputs, such as physi(4 35 r 30 -
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,
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27
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FIGURE 2. Cumulative number of eggs for cardboard and feather treatment during the oviposition cycle following the meal. (a) Animals were fed weekly and had a continuous oviposition activity previous to the beginning of the experiment. Data from Fig. l(a); (b) Animals were fasted 1 month (18 days of starvation) and had a sporadic or no oviposition before the experiment began. Data from Fig. l(b).
cal and/or chemical cues from the oviposition substrates, modulate the ovarian function. At least two different mechanisms could explain this modulation: (1) a modification in the metabolic conversion of nutrients into eggs (Table l), and (2) an inhibition of the growth of other oocytes by means of an antigonadotropic factor resulting from the retention of mature eggs [Fig. l(a, b)]. The more efficient conversion of the blood ingested in laid eggs observed for the fresh feather group (Table 1) can be explained indirectly by an increase in fecundity. Previous works of Nutiez and Segura (1987) demonstrated that in Triatoma infestans, the amount of eggs produced by a female is inversely proportional to the amount of blood invested to produce an egg. This fact indicates that the differences in blood conversion would be due to differences in the absolute fecundity between the experimental groups. The long-term assays (second series) revealed differences in the amount of eggs laid between cardboard and feather groups. Comparing Fig. l(a, b), we can see an early first peak of oviposition occurring during the first three days in the feather treatment [Fig. l(a)]. This was certainly due to the retention of mature eggs from the previous oviposition cycle (Chiang and Chiang, 1995). This early peak is absent in Fig. l(b), because of the lack or small number of eggs retained after 18 days of starvation. From the analysis of Fig. l(a, b), we conclude that there was no difference in the number of spermatophores, i.e. in the frequency of mating between experimental groups. Thus, the decrease in fecundity in the cardboard treatment cannot be explained by a reduction in the frequency of copula, but is only due to the absence of a feather substrate. A delay between the peaks of oviposition of both treatments of about 6 days could be observed in Fig. l(b). This delay would result from a transient retention of mature eggs in the ovary that, in turn, would inhibit further egg production by an antigonadotropic factor (Pratt and Davey, 1972a). Differences become more evident when the cumulative number of eggs is analysed [Fig. 2(a, b)]. These results match very well with those obtained by Davey (1967) working with virgin females, which retained mature eggs for a much longer time than mated females. In addition, our results from cardboard females resemble those of Buxton (1930) Coles (1965) and Davey (1965) in virgin females. These authors found that unmated R. prolixus lay about two-thirds the number of eggs produced by mated females. In our case the same decrease in fecundity [one-third, Fig. 2(a, b)] would be the result of the lack of an appropriate oviposition site, i.e. avian feather. These two analogies suggest the involvement of similar mechanisms of regulation of egg production or at least a common point in the pathway with two different inputs, i.e. adequate substrate for oviposition (our experiments) and copula (Pratt and Davey, 1972a).
SUBSTRATE-DEPENDENT
In addition, these results bring relevant information for rearing bugs in the laboratory: the presence of feathers in the rearing cages enhance the fecundity of R. prolixus. Besides, this modulation of the ovarian function depending on the availability of oviposition sites could explain changes in size of wild populations for the most important Chagas disease vector in the north of South America. Although our assays were made with R. prolixus, some evidence suggests that the results can be generalized to other Triatominae. The related species Triatoma infestans does not attach the eggs, but lays them inside crevices instead. Studies by Cecere et al. (1995) reveal that bugs living in experimental buildings differing in the availability of crevices display a differential fecundity, survival and density. REFERENCES Abu-hakima R. and Davey K. G. (1977) The action of juvenile hormone on the follicle cells of Rhodnius prolixus: the importance of volume changes. J. Exp. Biol. 69, 3344. Buxton P. A. (1930) The biology of a blood-sucking Rhodnius prolixus. Trans. R. Ent. Sot. Land. F8, 2277236. Cecere M. C., Canale D. and Gtirtler R. E. (1995) Efecto de la disponibihdad de refugios sobre la dinamica poblacional de Triatoma infestans en un modelo de ranchos experimentales: resultados parciales. Restimenes XVII Reunidn Argentina de Ecologia, Mar de1 Plats, pp. 85-86. Chiang R. G. and Chiang J. A. (1995) Fecundity of the blood-feeding insect Rhodniusprolixus increases in successive periods of egg production. Experientia 51, 2899292. Coles G. C. (1965) Haemolymph proteins and yolk proteins in Rhodnius prolixus Stal. J. Exp. Biol. 43, 42543 1.
FECUNDITY
IN R. PROLIXUS
841
Davey K. G. (1965) Copulation and egg-production in Rhodnius prolixus: the role of the spermatecae. J. Exp. Bioi. 42, 373-378. Davey K. G. (1967) Some consequences of copulation in Rhodnius prolixus. J. Insect Physiol. 13, 1629-1636. Davey K. G. and Kuster J. E. (I 98 I) The source of an antigonadotropin in the female of Rhodnius prolixus Stal. Can. J. Zool. 59, 761-764. Kriger F. L. and Davey K. G. (1982) Ovarian motility in mated Rhodnius prolixus requires an intact cerebral neurosecretory system. Gen. Camp. Endocrinol. 48, 130-134. Lent H. and Wygodzinsky P. (1979) Revision of the Triatominae (Hemiptera, Reduviidae), and their significance as vectors of Chagas’ disease. Bull. Am. Museum Nat. Hist. 163, article 3. NliAez J. A. and Lazzari C. R. (1990) Rearing of Triatoma infestans Klug (Het., Reduviidae) in the absence of a live host. I. Some factors affecting the artificial feeding. J. Appl. Entomol. 109, 87-92. Ntifiez J. A. and Segura E. L. (1987) Rearing of Triatominae. In Chagus’ Disease Vectors (Eds Brenner R. R. and Stoka A. M.), Vol. II, pp. 31-40. CRC Press, Boca Raton, Florida. Pratt G. E. and Davey K. G. (1972a) The corpus allatum and oogenesis in Rhodniusprolixus (St&). Ill. The effect of mating. J. Exp. Biol. 56, 223-237. Pratt G. E. and Davey K. G. (1972b) The corpus allatum and oogenesis in Rhodnius prolixus (Stal.). Il. The effects of starvation. J. Exp. Biol. 56, 215-221. Zar J. H. (1984) Biostutistical Analysis. Prentice-Hall, Inc., Englewood Cliffs, New Jersey.
Acknowledgements~We are indebted to M. Giurfa, W. Farina and G. Flares for critical reading of an early version of the manuscript, R. Gtirtler for many helpful discussions and H. Vema for providing the feathers. Special thanks are given to Dr K. Davey and two anonymous reviewers for many helpful suggestions. This investigation received financial support from the UNDPiWorld Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), CONICET and the Universidad de Buenos Aires/Argentina. Pablo Schilman was supported by a Doctoral grant from CONlCETiArgentina.
1996 J. Insect Physiol. Vol. 42, No. 9, pp. 837-841, Copyright 0 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910196 $15.00 + 0.00
Attributes of Oviposition Substrates Affect Fecundity in Rhodnius prolixus PABLO
E. SCHILMAN,*t
Received
8 November
JOSUh
A. NtJfdEZ,*
1995; revised and accepted
CLAUDIO
26 February
R. LAZZARI*
1996
The effect of the oviposition substrate on the rate of egg laying was investigated in the haematophagous bug Rhodnius prolixus. Fresh or old hen feathers or corrugated cardboard were offered to ovipositing females. The insects showed significant differences in the number of eggs laid depending on the substrate. Egg laying was maximal on the fresh feathers and minimal on the cardboard. The variations cannot be explained by differences in the amount of blood ingested nor by differences in the mating frequency between treatments. The amount of blood necessary per laid egg exhibited an inverse relationship with the absolute fecundity. The dynamics of,mating and egg-laying for groups having two different substrates (corrugated cardboard and hen feather) and different nutritional states (previously starved vs previously fed) was established throughout one month. After a single meal, previously starved insects having feathers as a substrate laid their eggs and reached an oviposition peak sooner (6 days) than previously starved insects on the cardboard substrate. For both nutritional states the lack of feathers decreased the cumulative number of eggs laid by one third. Results are discussed in relation to the mechanisms of ovarian regulation known at present. Copyright 0 1996 Elsevier Science Ltd R. prolixus
Mating
Fecundity
Oviposition
substrates
INTRODUCTION
The selection of suitable oviposition sites by female insects is of great importance to ensure that the eggs would be adequately protected from the environment and that food will be available for the larvae when they hatch. Triatominae exhibit two different strategies of oviposition: (1) species associated with birds attach their eggs to the feathers of their host; (2) other species lay their eggs in crevices (Lent and Wygodzinsky, 1979). Rhodnius prolixus, an important vector of Chagas’ disease in the north of South America and a classical model in insect physiology, belongs to the first group. In nature, this species inhabits birds nests, but it is also found in human housings due to passive transport or active displacement (Lent and Wygodzinsky, 1979). Feeding and mating are involved in the modulation of ovarian function in R. prolixus. As in most insects, the development of oocytes in this bug is under hormonal
*Laboratorio de Fisiologia de Insectos, Departamento de Cs. BioKgicas, Facultad de Cs. Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria (1428), Buenos Aires, Argentina. tTo whom all correspondence should be addressed. 837
Ovarian function
Chagas’
disease
control and involves cerebral neurosecretory cells and the corpora allata. In R. prolixus a batch of eggs is laid following each blood meal, and the number of eggs depends on the size of the meal ingested (Buxton, 1930). Feeding promotes the secretion of juvenile hormone, which regulates vitellogenin production by the fat body (Coles, 1965), previtellogenic development of the oocytes and vitellogenesis. In the latter case, the juvenile hormone induces the separation of the follicle cells from each other, so that haemolymph proteins have free access to the oocytes (Abu-Hakima and Davey, 1977). Mating, on the other hand, provokes the release of blood-borne substances from the spermathecae of the mated female, which stimulate the neurosecretory cells of the pars intercerebralis of the brain. As a response, these release a myotropic factor that increases the motility of the oviduct muscles (Kriger and Davey, 1982). The presence of mature eggs in the ovary causes peripheral neurosecretory cells to secrete an antigonadotropic factor, which blocks vitellogenesis in younger oocytes (Davey and Kuster, 1981). In this report, we analyse the effect of an external input other than mating and feeding, i.e. the availability of different substrates for oviposition on the ovarian function of R. prolixus. In a first series of experiments, different
PABLO E. SCHILMAN et al.
838
substrates were offered to females of R. prolixus and changes in the fecundity (egg laying) and blood-egg conversion were analysed. In a second series of experiments, the dynamics of copulation and oviposition under the different treatments was studied. MATERIALS AND METHODS Adults of R. prolixus, 2-3 months old, reared in the laboratory at 28°C and constant dark regime, were used throughout. The insects were fed weekly with heparinized bovine blood using an artificial feeder (Nuiiez and Lazzari, 1990) as nymphs and adults. Adults were maintained in plastic recipients of 500 ml capacity in groups of about 15 females and 15 males. A piece of corrugated cardboard and hen feathers were present inside each recipient. At the beginning of the experiments, females were mated and had been through about 8-l 2 oviposition cycles. Insects were distributed in 15 equal sized compartments (5.5 x 5.5 x 4.5 cm) of a transparent acrylic box of 22.5 x 17.5 x 4.5 cm. The acrylic cover was perforated to allow air circulation. The floor was coated with filter paper. The acrylic box was kept at 28°C under a constant dark regime. First experimental series In this series, the fecundity (number of eggs laid) of females in the presence of different oviposition substrates was analysed. Females were marked with acrylic paint for identification and individually weighed to the nearest 0.1 mg. They were placed in a communal jar for feeding for two consecutive days (afternoon and next morning) to ensure feeding of a high proportion of insects. Three hours after the second meal, they were weighed again and the amount of blood ingested by each insect‘calculated. Male insects were fed at the same time as the females in a different communal jar. Thereafter, groups of one female and two males were put together in each compartment with a given substrate for oviposition (day 0). Three different substrates were offered: (1) a piece of corrugated cardboard and a feather removed from a hen within 24h before the beginning of the experiment (henceforth: fresh feather); (2) a piece of corrugated cardboard and a feather removed from a hen at least one month before the beginning of the experiment (henceforth: old feather); and (3) two pieces of corrugated cardboard. The feathers were wrapped with aluminium foil and kept until the experiment began. Each treatment lasted 1 week and was repeated in three successive weeks changing the oviposition substrate, so that every female was exposed to all different substrates avoiding individual variation. The chambers were washed between treatments. Insects were fed once a week as described and the eggs laid during the time they were in the communal feeding jar were not considered in the calculations. Number of eggs laid per week as related to substrates
for oviposition (fresh feather, old feather and cardboard) was statistically analysed by means of nonparametric randomized block analysis of variance (Friedman test). Multiple comparisons between groups were performed by a posteriori Dunn’s test (Zar, 1984). Two replications of this experimental series were made (n = 15 females and 30 males) involving a total number of 30 females and 60 males. Females that did not feed were excluded from the analysis to avoid any increase in variability by a factor (feeding) that must be under control. As a consequence, the initial number of females used (30) was reduced to 12. Second experimental series The dynamics of egg laying and mating was analysed for two different substrates (cardboard and fresh feather) and for insects differing in their nutritional state. Two groups of 15 mated females and 30 males were used, each differing in its nutritional history. One group was fed weekly since the imaginal ecdysis and displayed a continuous oviposition behaviour for about 8-l 2 weeks before the experiment began. The other group was not fed for one month and showed sporadic oviposition previous to the beginning of the experiment. Bugs belonging to the latter group had about 18 days of true starvation. This is due to the fact that R. prolixus takes a large blood meal which is stored in the crop and animals can only be considered as ‘starved’ when these reserves are exhausted. In a mated female, this occurs in the 12th day after feeding (Pratt and Davey, 1972b). The two nutritional states used in this experiment are thus equivalent to 0 and 18 days of starvation. In this series, we analysed the incidence of the characteristics of the substrate and nutritional state in the dynamics of egg laying and mating. Insects were fed once at the beginning (day 0), as described in the first series, and not fed until the end of the experiment (day 30). Two replications of each assay were made. In the group fed weekly prior to the beginning of the experiment, a total of 30 females (15 cardboard + 15 feather) and 60 males were used. At the end of the experimental period (1 month), 28 females of this group were taken into account, because two cardboard insects died. Different insects were used in each replication. In the ‘starved’ group, 15 females and 30 males were observed throughout one month. Thereafter the insects were fed, groups interchanged (cardboard and feather) and the insects tested for another 30 days. During the course of the experiment, 1 feather and 2 cardboard insects died. The number of eggs laid by each female and the spermatophores found in each compartment were recorded every 3 days. Data were statistically analysed by means of a twofactor analysis of variance with equal replication.
SUBSTRATE-DEPENDENT
FECUNDITY
IN R. PROLIXUS
839
RESULTS First experimental
series
The number of eggs laid was significantly different depending on the oviposition substrate available (Table 1). The number of eggs laid by the cardboard group differed significantly from that of the fresh feather group and old feather group. No significant difference was found between old and fresh feather groups. The differences in the amount of eggs laid were independent of the amount of blood ingested (Table 1) and were therefore due to the nature of the substrate offered. Differences in the conversion of blood ingested into eggs laid were evinced between groups (Table 1). As the ratio was calculated from total amounts of laid eggs and blood ingested by the 12 females, no standard errors were obtained and, therefore, no statistical analysis could be applied. The reason for this procedure was the fact that although all the females ate, not all of them oviposited. Second experimental
series
In the long-term assay, the number of spermatophores and the number of eggs laid were analysed over a month (Fig. 1). Concerning egg laying, ANOVA analysis revealed significant effects of the treatments (cardboard vs feather, F = 7.284, df = 1, P = O.Ol), but not of the previous nutritional status (weekly fed vs starved, F = 2.418, df = 1, P = 0.127), nor of the interaction between factors (treatment and nutritional status, F = 0.352, df = 1, P = 0.556). Differences within groups were revealed as a function of time (days of experiment, F = 4.028, df = 8, P
TABLE
1. Relationship
between
blood
Treatment
Females
Cardboard Old feather Fresh feather Friedman test
12 12 12
(n)
ingestion
0
3
6
9
12
15
Days after feeding FIGURE 1. The number of eggs laid (bar plot; x + SE) and the number of spermatophores (line plot; R ? SE) for both substrate treatments (cardboard and feather) as related to the time after feeding. The sampling interval was 3 days. (a) animals were fed weekly and had a continuous oviposition activity previous to the beginning of the experiment; (b) animals were fasted for 1 month (18 days of starvation) and had a sporadic or no oviposition before the experiment began.
Statistical analysis (ANOVA) revealed differences in the dynamics according to both treatments (F = 3.920, df = 8, P
and egg production by R. oviposition
prolixus females and the availability
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Meal size (mg)
Eggs laid
mg blood/egg
37.78k4.19 49.15f7.74 47.92H4.21 ,+4.979, df=2, ns
lf0.37 10.67ti.10 17.25+3.19 ,$=11.787,
31.76 4.61 2.18
substrates
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df=2, p
Significant differences in the number of eggs laid but not in the meal size were found among treatments “Dunn’s test a posteriori (df = 3) fresh feather vs cardboard, Q = 3.866, P
Q = 2.762, P cO.05;
old vs
PABLO E. SCHILMAN et al.
840
Although the fertility of the eggs was not quantified, most of them hatched in both groups and treatments, indicating that a successful sperm transference took place. The analysis of the dynamics of accumulated eggs [Fig. 2(a, b)] reveals that cardboard females laid about one-third less eggs than the feather females. A significant difference was found between cardboard and feather in both nutritional states [Fig. 2(a): t = 2.197, df = 26, P CO.05 and Fig. 2(b): t = 2.359, df = 25, P cO.051. No significant difference was found between cardboard and feather treatments in the amount of blood ingested in both nutritional states (unpaired t-test, data not shown). DISCUSSION
Up to now, the mechanisms controlling egg production have been analysed in terms of internal regulation paths (i.e. nervous, endocrine), modulated by mating and feeding. Our results show that external inputs, such as physi(4 35 r 30 -
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FIGURE 2. Cumulative number of eggs for cardboard and feather treatment during the oviposition cycle following the meal. (a) Animals were fed weekly and had a continuous oviposition activity previous to the beginning of the experiment. Data from Fig. l(a); (b) Animals were fasted 1 month (18 days of starvation) and had a sporadic or no oviposition before the experiment began. Data from Fig. l(b).
cal and/or chemical cues from the oviposition substrates, modulate the ovarian function. At least two different mechanisms could explain this modulation: (1) a modification in the metabolic conversion of nutrients into eggs (Table l), and (2) an inhibition of the growth of other oocytes by means of an antigonadotropic factor resulting from the retention of mature eggs [Fig. l(a, b)]. The more efficient conversion of the blood ingested in laid eggs observed for the fresh feather group (Table 1) can be explained indirectly by an increase in fecundity. Previous works of Nutiez and Segura (1987) demonstrated that in Triatoma infestans, the amount of eggs produced by a female is inversely proportional to the amount of blood invested to produce an egg. This fact indicates that the differences in blood conversion would be due to differences in the absolute fecundity between the experimental groups. The long-term assays (second series) revealed differences in the amount of eggs laid between cardboard and feather groups. Comparing Fig. l(a, b), we can see an early first peak of oviposition occurring during the first three days in the feather treatment [Fig. l(a)]. This was certainly due to the retention of mature eggs from the previous oviposition cycle (Chiang and Chiang, 1995). This early peak is absent in Fig. l(b), because of the lack or small number of eggs retained after 18 days of starvation. From the analysis of Fig. l(a, b), we conclude that there was no difference in the number of spermatophores, i.e. in the frequency of mating between experimental groups. Thus, the decrease in fecundity in the cardboard treatment cannot be explained by a reduction in the frequency of copula, but is only due to the absence of a feather substrate. A delay between the peaks of oviposition of both treatments of about 6 days could be observed in Fig. l(b). This delay would result from a transient retention of mature eggs in the ovary that, in turn, would inhibit further egg production by an antigonadotropic factor (Pratt and Davey, 1972a). Differences become more evident when the cumulative number of eggs is analysed [Fig. 2(a, b)]. These results match very well with those obtained by Davey (1967) working with virgin females, which retained mature eggs for a much longer time than mated females. In addition, our results from cardboard females resemble those of Buxton (1930) Coles (1965) and Davey (1965) in virgin females. These authors found that unmated R. prolixus lay about two-thirds the number of eggs produced by mated females. In our case the same decrease in fecundity [one-third, Fig. 2(a, b)] would be the result of the lack of an appropriate oviposition site, i.e. avian feather. These two analogies suggest the involvement of similar mechanisms of regulation of egg production or at least a common point in the pathway with two different inputs, i.e. adequate substrate for oviposition (our experiments) and copula (Pratt and Davey, 1972a).
SUBSTRATE-DEPENDENT
In addition, these results bring relevant information for rearing bugs in the laboratory: the presence of feathers in the rearing cages enhance the fecundity of R. prolixus. Besides, this modulation of the ovarian function depending on the availability of oviposition sites could explain changes in size of wild populations for the most important Chagas disease vector in the north of South America. Although our assays were made with R. prolixus, some evidence suggests that the results can be generalized to other Triatominae. The related species Triatoma infestans does not attach the eggs, but lays them inside crevices instead. Studies by Cecere et al. (1995) reveal that bugs living in experimental buildings differing in the availability of crevices display a differential fecundity, survival and density. REFERENCES Abu-hakima R. and Davey K. G. (1977) The action of juvenile hormone on the follicle cells of Rhodnius prolixus: the importance of volume changes. J. Exp. Biol. 69, 3344. Buxton P. A. (1930) The biology of a blood-sucking Rhodnius prolixus. Trans. R. Ent. Sot. Land. F8, 2277236. Cecere M. C., Canale D. and Gtirtler R. E. (1995) Efecto de la disponibihdad de refugios sobre la dinamica poblacional de Triatoma infestans en un modelo de ranchos experimentales: resultados parciales. Restimenes XVII Reunidn Argentina de Ecologia, Mar de1 Plats, pp. 85-86. Chiang R. G. and Chiang J. A. (1995) Fecundity of the blood-feeding insect Rhodniusprolixus increases in successive periods of egg production. Experientia 51, 2899292. Coles G. C. (1965) Haemolymph proteins and yolk proteins in Rhodnius prolixus Stal. J. Exp. Biol. 43, 42543 1.
FECUNDITY
IN R. PROLIXUS
841
Davey K. G. (1965) Copulation and egg-production in Rhodnius prolixus: the role of the spermatecae. J. Exp. Bioi. 42, 373-378. Davey K. G. (1967) Some consequences of copulation in Rhodnius prolixus. J. Insect Physiol. 13, 1629-1636. Davey K. G. and Kuster J. E. (I 98 I) The source of an antigonadotropin in the female of Rhodnius prolixus Stal. Can. J. Zool. 59, 761-764. Kriger F. L. and Davey K. G. (1982) Ovarian motility in mated Rhodnius prolixus requires an intact cerebral neurosecretory system. Gen. Camp. Endocrinol. 48, 130-134. Lent H. and Wygodzinsky P. (1979) Revision of the Triatominae (Hemiptera, Reduviidae), and their significance as vectors of Chagas’ disease. Bull. Am. Museum Nat. Hist. 163, article 3. NliAez J. A. and Lazzari C. R. (1990) Rearing of Triatoma infestans Klug (Het., Reduviidae) in the absence of a live host. I. Some factors affecting the artificial feeding. J. Appl. Entomol. 109, 87-92. Ntifiez J. A. and Segura E. L. (1987) Rearing of Triatominae. In Chagus’ Disease Vectors (Eds Brenner R. R. and Stoka A. M.), Vol. II, pp. 31-40. CRC Press, Boca Raton, Florida. Pratt G. E. and Davey K. G. (1972a) The corpus allatum and oogenesis in Rhodniusprolixus (St&). Ill. The effect of mating. J. Exp. Biol. 56, 223-237. Pratt G. E. and Davey K. G. (1972b) The corpus allatum and oogenesis in Rhodnius prolixus (Stal.). Il. The effects of starvation. J. Exp. Biol. 56, 215-221. Zar J. H. (1984) Biostutistical Analysis. Prentice-Hall, Inc., Englewood Cliffs, New Jersey.
Acknowledgements~We are indebted to M. Giurfa, W. Farina and G. Flares for critical reading of an early version of the manuscript, R. Gtirtler for many helpful discussions and H. Vema for providing the feathers. Special thanks are given to Dr K. Davey and two anonymous reviewers for many helpful suggestions. This investigation received financial support from the UNDPiWorld Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), CONICET and the Universidad de Buenos Aires/Argentina. Pablo Schilman was supported by a Doctoral grant from CONlCETiArgentina.