Research Article |
Corresponding author: Pablo Liedo ( jvalle@ecosur.mx ) Academic editor: Gavin Broad
© 2020 Jassmin Cruz-Bustos, Pablo Montoya, Gabriela Pérez-Lachaud, Javier Valle-Mora, Pablo Liedo.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Cruz-Bustos J, Montoya P, Pérez-Lachaud G, Valle-Mora J, Liedo P (2020) Biological attributes of diapausing and non-diapausing Doryctobracon areolatus (Hymenoptera, Braconidae), a parasitoid of Anastrepha spp. (Diptera, Tephritidae) fruit flies. Journal of Hymenoptera Research 78: 41-56. https://doi.org/10.3897/jhr.78.52269
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Doryctobracon areolatus (Szépligeti), a solitary endoparasitoid native to the Neotropics, attacks eggs and early instar larvae of Anastrepha fruit flies, and can enter diapause under tropical and subtropical conditions. We aimed to test if biological attributes, such as size, flight ability, starvation resistance, longevity and fecundity of diapausing individuals differ from those of non-diapausing ones. Parasitoids were obtained from a laboratory colony reared on Anastrepha ludens (Loew) larvae. Parasitized host puparia were sorted in two cohorts according to their diapause condition. Developmental time from egg to adult ranged from 18 to 31 days in non-diapausing parasitoids, and 70 to 278 days for diapausing individuals. Pupal weight and adult measurements were higher in non-diapausing than in diapausing parasitoids. There were no differences in adult longevity, starvation resistance, and emergence between diapausing and non-diapausing wasps. Flight ability and fecundity rates were greater in the non-diapausing than in the diapause cohort. The proportion of female offspring was greater in the non-diapausing cohort (42.5%), whereas in the diapausing cohort the male offspring proportion was greater (62.4%). Both cohorts produced diapause offspring, but the non-diapausing cohort produced more (26.6%) than the diapausing one (9.1%). Maternal age had a significant effect on the proportion of diapause offspring: in 26 to 34 days old non-diapausing females, 78.9% of their offspring entered into diapause. These results confirmed that diapause affects the biological attributes of D. areolatus. The observed differences contribute to better understand the diapause influence on the colonization and rearing process of this species and its use as biocontrol agent.
Braconidae, fecundity, flight ability, size, Summer diapause, survival, Tephritidae
Tephritid fruit fly parasitoids are grouped in five families of Hymenoptera: Braconidae, Diapriidae, Eulophidae, Figitidae, and Ichneumonidae (
Diapause is defined as a type of dormancy in which metabolic and developmental arrest occur in the life cycles of many invertebrates (
The genus Doryctobracon Enderlein, 1920, is endemic to the Americas (
Doryctobracon areolatus developmental time, when it does not diapause, is 27 days (
The study of diapause in fruit fly parasitoids is relevant not only because of its potential to infer evolutionary relationships, but also to provide insights into the use of these species as biocontrol agents. Biological characteristics of diapausing individuals such as reproductive capacity, tolerance to environmental stress or flight ability can influence their use in biological control projects (
Our aim in this research was to determine if there are differences in Doryctobracon areolatus between biological attributes (size, flying ability, starvation resistance, longevity, fecundity, offspring sex ratio and diapause frequency) of individuals which diapause and directly developing individuals, to infer if this condition influences the fitness of this species, and the implications for its mass rearing.
Parasitized host puparia and adult parasitoids came from the D. areolatus colony that is maintained in the Biological Control laboratory of the Moscafrut Program (SADER-SENASICA), located in Metapa, Chiapas, Mexico, which at the time of bioassays had 23 generations under laboratory rearing conditions. Second instar larvae of A. ludens were used as hosts. Puparia were placed in 30 ml plastic containers, covered with organza fabric to allow ventilation. They were kept on a coconut fiber substrate that was kept slightly humid with water applied by spraying until adult emergence. Laboratory conditions were 24 ± 1 °C temperature, 80–90% relative humidity and a 12:12 L:D cycle.
Two cohorts were obtained based on the type of development of the parasitoids: host puparia with parasitoids without diapause (direct development), and host puparia with evidence of parasitoids in diapause (hereafter non-diapausing and diapausing, respectively). The puparia containing larvae of diapausing parasitoids were distinguished by observing the 3rd instar parasitoid larva inside the host pupa under a stereomicroscope. We recorded the developmental time for both cohorts.
Fly puparia were individually weighed using an analytical scale (Ohaus, Pine Brook, NJ) and then placed in plastic containers with 24 independent cells. Each cell was conditioned with lightly moistened coconut fiber substrate, where they remained under laboratory conditions until adult emergence.
Pupal and adult measurements were made with a stereomicroscope (Carl Zeiss, Stemi 2000C) fitted with a scale in the right eyepiece. Thirty host puparia containing parasitoid larvae of each physiological condition were randomly selected and the width and length, from the end of the buccal carinae to the end of the anal pore, were measured. For adult measurements, the cells were checked daily, recording for each emerged parasitoid the date and sex. Each individual was placed in a 1.5 ml vial with an 80% alcohol solution. We measured: 1) length of the left posterior tibia, 2) length of the left wing, 3) mesosoma length, 4) metasoma length, 5) antenna length, and 6) ovipositor length (
Samples of 100 diapausing and 100 non-diapausing pupae were placed inside a 10 cm diameter X 10 cm height PVC tube, painted black, with the inner wall of the tube covered with neutral talcum powder to prevent the outflow of non-flying parasitoids (as in
Adults that emerged from both diapausing and non-diapausing larvae were individually placed in 10×12×16 cm plastic cages. Honey embedded in towel paper placed on a plastic lid (1.5 × 0.07 cm) was provided as food (
For starvation resistance, at emergence, 30 males and 30 females for each type of development (direct development and diapause) were placed in plastic cages (10×12×16cm) without food and water. Daily, the cages were checked and the dead parasitoids were collected and recorded, noting their type of development, sex and age.
Forty pairs of adults emerged from both diapausing and non-diapausing larvae were used. Each pair was placed in a 10×12×16 cm plastic cage. The individuals were provided with food (honey) and water as described above. The food was changed twice a week. To evaluate fecundity we used artificial oviposition units made of a guava fruit (Psidium guajava L.) (Myrtaceae) (
Anastrepha ludens larvae were removed from the oviposition devices and placed in containers with larval diet for seven more days. Then, the mature larvae were separated from the food with a sieve and water and placed in plastic bottles with moist coconut fiber to promote pupation. At emergence, the number and sex of the emerged adult parasitoids were recorded. In the case of non-emerged pupae, they were examined under a stereomicroscope to determine if they were in diapause, dead or were unparasitized A. ludens pupae. For each female we recorded the number of offspring produced per day, noting males, females and diapausing individuals.
Differences in developmental time (mean ± SE) were analyzed by a t test. Morphometric data (mean ± SE) were analyzed using a canonical multivariate analysis of variance (MANOVA) (
A total of 5,832 host puparia with evidence of diapausing parasitoids, and 934 puparia with non diapausing parasitoids were used in the bioassays. Developmental time from egg to adult, which was from the exposure of the host (A. ludens second instar larvae) to adult emergence, ranged from 70 to 278 days for diapausing parasitoids and from 18 to 31 days in non-diapausing ones (Fig.
Developmental time and morphological measurements of non-diapausing and diapausing Doryctobracon areolatus parasitoids and host puparia. SE: Standard error, n = sample size. Values followed by different letters in each row are significantly different (P < 0.05, canonical discriminant analysis).
Type of development | Non-diapausing | Diapause | ||||||
---|---|---|---|---|---|---|---|---|
Sex | ♂ | ♀ | ♂ | ♀ | ||||
Parameter | Mean ± SE | n | Mean ± SE | n | Mean ± SE | n | Mean ± SE | n |
Development time (days) | 21.86 ± 0.16 c | 134 | 23.35 ± 0.15 b | 179 | 191.60 ± 1.62 a | 384 | 188.85 ± 1.76 a | 317 |
Puparia weight (mg) | 13.1 ± 0.5 ab | 29 | 15.0 ± 0.6 c | 37 | 12.0 ± 0.4 ab | 32 | 14.0 ± 0.5 a | 26 |
Puparia length (mm) | 6.25 ± 0.15 ab | 29 | 6.39 ± 0.09 c | 37 | 6.24 ± 0.06 ab | 32 | 6.52 ± 0.08 a | 26 |
Puparia width (mm) | 2.84 ± 0.03 a | 29 | 2.88 ± 0.04 a | 37 | 2.81 ± 0.03 a | 31 | 2.92 ± 0.04 a | 24 |
Adults | ||||||||
Tibia length (mm) | 1.53 ± 0.02 a | 27 | 1.56 ± 0.02 a | 28 | 1.46 ± 0.03 b | 13 | 1.52 ± 0.02 a | 14 |
Wing length (mm) | 4.88 ± 0.04 b | 27 | 5.13 ± 0.08 a | 28 | 4.48 ± 0.07 c | 13 | 4.89 ± 0.04 b | 14 |
Thorax length (mm) | 2.03 ± 0.03 a | 27 | 2.13 ± 0.04 a | 28 | 1.99 ± 0.03 a | 13 | 2.29 ± 0.24 a | 14 |
Abdomen length (mm) | 3.37 ± 0.07 a | 27 | 3.30 ± 0.06 a | 28 | 3.31 ± 0.07 a | 13 | 3.09 ± 0.09 b | 14 |
Antenna length (mm) | 7.64 ± 0.10 a | 27 | 7.27 ± 0.10 b | 28 | 7.08 ± 0.10 b | 13 | 6.64 ± 0.11 c | 14 |
Ovipositor length (mm) | – | 4.92 ± 0.07 a | 28 | – | 4.69 ± 0.24 b | 14 |
The multivariate canonical analysis, considering the length, width and weight of the host puparia containing wasps in diapause and wasps without diapause, indicated a significant interaction of sex and type of development (Manova, F3,115 = 3.86, P < 0.01). The host puparium weight of non-diapausing parasitoids was greater than that of diapausing ones. However, host puparia of diapausing female parasitoids tended to be longer and wider, but the only significant difference was in puparium length when compared with non-diapausing females. Likewise, host puparia from which female parasitoids emerged were heavier, longer and wider than the puparia containing males (Table
Regarding parasitoid size, statistical differences were found in the type of development (F5, 74 = 7.06, P < 0.0001) and sex (F5, 74 = 17.78, P < 0.0001), but there was not a significant interaction between these two factors (F5, 74 = 1.07, P > 0.05). Parasitoids directly developing had longer tibia, wing, abdomen, and antenna, compared to parasitoids that diapaused (Table
Adult emergence rate was higher in non-diapausing parasitoids (75.69% ± 2.96) than in diapausing parasitoids (39.86% ± 9.69). However, the difference was not statistically significant (Student t test, t = 2.82 df = 3, P > 0.05). The percentage of flying parasitoids from direct development (55.56% ± 3.54) was significantly higher than in diapausing parasitoids (23.36% ± 5.36) (t = 4.03, df = 3, P < 0.05) (Table
Flight ability and emergence rate of non-diapausing and diapausing Doryctobracon areolatus parasitoids. Emergence rate is the proportion of adults emerged from 100 host puparia. Percentage of flyers is the proportion of parasitoids capable of flying from 100 host puparia. SE = Standard Error. Values followed by different letters in each column are significantly different (t = 4.03, df = 3, P < 0.05).
Cohort | Replicates | Emergence rate (%) ± SE | Fliers (%) ± SE |
---|---|---|---|
Non-Diapausing | 2 | 75.69 ± 2.96 a | 55.56 ± 3.54 a |
Diapausing | 3 | 39.86 ± 9.69 a | 23.36 ± 5.36 b |
There were significant differences in survival between starved individuals and those provided with food (Log-rank test, χ2 = 98.46, df = 7, P < 0.001). When food was provided, diapausing females showed the greatest mean longevity (24 days, Table
Mean longevity (± SE) and starvation resistance (in days) in non-diapausing and diapausing Doryctobracon areolatus parasitoids.
Sex | Type of development | Longevity (days) | |||
---|---|---|---|---|---|
With food | n | Without food | n | ||
♀ | Non-diapausing | 14.77 ± 2.27 ab | 43 | 4.18 ± 0.33 a | 55 |
Diapausing | 24.00 ± 3.04 a | 30 | 5.40 ± 0.27 a | 45 | |
♂ | Non-diapausing | 12.09 ± 1.48 ab | 47 | 4.40 ± 0.40 a | 43 |
Diapausing | 10.67 ± 1.35 b | 52 | 4.33 ± 0.24 a | 52 |
No significant differences were found in starvation resistance between diapausing and non-diapausing cohorts, nor between females and males (Table
In the fecundity bioassays when females were provided with hosts, there was no difference in the survival of the females of both conditions (Z = 1.24, P > 0.05, Fig.
Differences in the reproduction of parasitoids emerged from direct development and diapause were observed, both in the fecundity rates and in the allocation of the offspring. Fecundity was higher for the non-diapausing cohort than for the diapausing one. Non-diapausing females also produced more daughters than diapausing ones and more individuals entering into diapause (Table
Fecundity rates (female offspring per female) of non-diapausing and diapausing Doryctobracon areolatus parasitoids and fraction of females, males and diapausing offspring.
Non-diapausing | Diapausing | |
---|---|---|
Gross fecundity (daughters / female) | 19.99 | 10.92 |
Net fecundity (daughters / female) | 8.47 | 8.00 |
Male offspring (%) | 30.87 | 62.54 |
Female offspring (%) | 42.58 | 28.49 |
Diapausing offspring (%) | 26.55 | 9.12 |
About 67% of both diapausing and non-diapausing pairs produced offspring. Of those pairs with offspring, 86.2% of the non-diapausing cohort and 100% of the diapausing cohort produced males, whereas 62% and 48%, respectively, produced females. There were no differences in the number of non-diapausing and diapausing pairs that produced daughters (χ2 = 1.07, P > 0.05). The fraction of pairs that produced offspring that entered into diapause was 58% and 40% for the non-diapausing, and diapausing cohorts, respectively. Maternal age in the non-diapausing cohort had an important effect on the production of offspring that entered into diapause. Over 78% of the offspring of 26 to 34 days-old females from the direct development condition entered into diapause, and the rest were only males (Fig.
Diapausing and non-diapausing D. areolatus individuals differed in pupal and adult size, flight ability, fecundity, and reproductive dynamics, whereas there were no differences in their percent of adult emergence, starvation resistance and adult survival when provided with food. Interestingly, females from both cohorts produced diapausing offspring. However, non-diapausing females produced a higher percentage of diapausing offspring than diapausing females, particularly at old ages (26 to 34 days-old). The physiological, behavioral and evolutionary reasons for this are new research questions.
Under our laboratory-controlled conditions and using mass-reared A. ludens as hosts, we found that diapause in D. areolatus has been maintained after 23 generations, suggesting a genetic component. The developmental time of diapausing individuals ranged from 70 to 281 days, which is shorter than those reported for this species when they were collected in the field in Mexico, Brazil and Argentina (
Another factor affecting diapause duration is the metabolic reserves of individuals (
It is generally agreed that diapause represents a strategy to cope with adverse environmental conditions, including a shortage of hosts. In this context, and contrary to the expectation of an increased starvation resistance in diapausing individuals, our results showed that diapausing and non-diapausing parasitoids had similar survival rates when deprived of food. However, females emerging from diapause and provided with food and water lived longer than non-diapausing females (Table
The higher proportion of female offspring produced by non-diapausing females could be attributed to a lower mating rate in the diapause cohort, thus increasing the production of parthenogenetic male individuals. The higher fecundity of the non-diapausing cohort and the sex ratio biased to females indicate that these parasitoids will show greater population growth rates than diapausing ones. This decrease in reproduction may represent a trade-off for diapause affecting the adult fitness components (
Our most important finding was the effect of mother age of non-diapausing females on the production of offspring entering into diapause. Females of 26 to 34 days old, produced 78% of the offspring that entered into diapause. This suggests that diapause in D. areolatus has a hard-wired genetic component and may represent an adaptation to host scarcity in the field (
We concluded that diapause in D. areolatus has effects on some biological attributes that can be trade-offs in the parasitoids fitness, such as pupal and adult size, flight ability and reproduction. We also found that maternal age has an important effect on the proportion of offspring entering diapause. This represents baseline knowledge to understand how diapause affects the population dynamics of this species and the possible implications in the implementation of the mass rearing and biocontrol applications. The presence of diapausing individuals as part of the offspring of the released population could improve the effectiveness of biocontrol strategies during unfavorable environmental conditions emerging at the optimal time.
We thank Jorge Cancino, Patricia Lopez, Amanda Ayala, Florida Lopez, Erick Flores, Patricia Rosario, Velisario Rivera, Cesar Perez and Cesar Galvez for their technical assistance. The Laboratorio de Control Biológico, Subdirección de Desarrollo de Métodos, Programa Moscafrut SADER-SENASICA (Mosacafrut) for the use of their facilities and their support. This research was a partial requirement for the Master in Science degree of Jassmin Cruz-Bustos at El Colegio de la Frontera Sur (ECOSUR) and was supported by a scholarship from the Consejo Nacional de Ciencia y Tecnología (CONACYT CVU 711462). The authors state that no conflict of interests exists.