Table of Contents Author Guidelines Submit a Manuscript
Volume 2017 (2017), Article ID 3156534, 8 pages
Research Article

An Insight in the Reproductive Biology of Therophilus javanus (Hymenoptera, Braconidae, and Agathidinae), a Potential Biological Control Agent against the Legume Pod Borer (Lepidoptera, Crambidae)

1UMR DGIMI 1333 INRA, UM, Case Courrier 101, Place Eugène Bataillon, 34 095 Montpellier, France
2International Institute of Tropical Agriculture, Benin Research Station (IITA-Benin), 08 BP 0932 Cotonou, Benin
3Faculté des Sciences Agronomiques (FSA), Université d’Abomey Calavi, 01 BP 526 Cotonou, Benin
4Department of Plant Sciences, Wageningen University & Research, Wageningen, Netherlands
5Department of Entomology, Michigan State University (MSU), East Lansing, MI, USA

Correspondence should be addressed to Anne-Nathalie Volkoff and Manuele Tamò

Received 23 June 2017; Accepted 17 August 2017; Published 28 September 2017

Academic Editor: Jacques Hubert Charles Delabie

Copyright © 2017 Djibril Aboubakar Souna et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Therophilus javanus is a koinobiont, solitary larval endoparasitoid currently being considered as a biological control agent against the pod borer Maruca vitrata, a devastating cowpea pest causing 20–80% crop losses in West Africa. We investigated ovary morphology and anatomy, oogenesis, potential fecundity, and egg load in T. javanus, as well as the effect of factors such as age of the female and parasitoid/host size at oviposition on egg load. The number of ovarioles was found to be variable and significantly influenced by the age/size of the M. vitrata caterpillar when parasitized. Egg load also was strongly influenced by both the instar of M. vitrata caterpillar at the moment of parasitism and wasp age. The practical implications of these findings for improving mass rearing of the parasitoid toward successful biological control of M. vitrata are discussed.

1. Introduction

The legume pod borer Maruca vitrata (Fabricius) (syn. M. testulalis) (Lepidoptera: Crambidae) is an important pest of cowpea, Vigna unguiculata L. Walp (Fabales: Fabaceae), a widely cultivated legume crop in Sub-Saharan Africa, and can cause yield losses in the range of 20–80% [1]. According to taxonomic and population genetic studies, the putative area of origin of this pest is assigned to South Asia [2]. Therophilus javanus (Bhat & Gupta, 1977) (Hymenoptera: Braconidae) is an endoparasitoid that develops inside M. vitrata during the early larval stages. High parasitism rates of T. javanus on M. vitrata caterpillars have been reported in soybean and yard-long beans fields in tropical Asia, Lao PDR, Vietnam, and Southern Taiwan [3, 4]. T. javanus thus seems an excellent candidate for use as a biological control agent against M. vitrata in West Africa. However, the development of biological control relying on T. javanus releases requires a thorough knowledge of its basic biology, which has not been investigated yet.

Therophilus javanus belongs to the Agathidinae subfamily of Braconidae, which includes an estimated two to three thousand species worldwide with a higher number of genera in tropical than in temperate regions [5]. Some species have been employed as biological control agents against various insect pests [6, 7]. Although Agathidinae have been studied for taxonomic or phylogeny purposes, the biology of members of this subfamily remains largely unknown. A few biological and quite ancient studies have been conducted on Agathidinae oviposition and larval development [811]. Most studied Agathidinae species oviposit into special organs (nerve ganglia) [12], but some, including T. javanus, place their eggs directly into the host hemocoel [810]. Apart from a few studies, that is, the number of eggs laid by Bracon vulgaris (Hymenoptera: Braconidae) [11], and the number of offspring produced by Agathis gibbosa (Hymenoptera: Braconidae) [9], there is a dearth of data concerning Agathidinae reproductive biology.

Fecundity is one of the proxies used by biologists to investigate the individual fitness [13] and may greatly vary depending on the species and its life cycle. It can also be affected by a series of abiotic (e.g., temperature) and biotic (e.g., wasp nutritional status, mating status, and age) parameters. Fecundity has been shown to correlate positively with the number of ovarioles, that is, the egg-producing components of the ovary [14]. The number of ovarioles is commonly species-specific, and there is great variation across insects, ranging from less than five per ovary in some flies to hundreds per ovary in some grasshoppers [15]. As such, ovariole number is relatively stable for a given species but can vary due to different environmental or nutritional conditions [16]. In some parasitoid species, adults emerge with their full load of mature eggs (termed “proovigenic”) while other species mature eggs during their adult life (termed “synovigenic”) [17]. For the latter, production of the first eggs relates to the amount of nutritional resources stored during larval stages [18, 19].

In the present work, we investigated T. javanus reproductive biology, ovariole number, egg development, and the potential fecundity, as well as how egg loads vary depending on the wasp female age and how they are affected by parameters such as the nutritional quality provided by the lepidopteran host (i.e., caterpillar instar).

2. Materials and Methods

2.1. Insect Rearing

Therophilus javanus was provided by The World Vegetable Center (AVRDC), Taiwan, Republic of China, and reared for several generations under confined conditions at IITA, Benin research station. The pod borer M. vitrata colony was established from feral populations collected from both cowpea fields and alternative host plants surrounding the IITA-Benin station. Insect colonies were reared under laboratory conditions, with 12 : 12 L : D photoperiod 26°C ± 1.1°C average temperature and 76%  ± 7% relative humidity. Four-day-old, mated adult females of M. vitrata were transferred in groups of four or five individuals to transparent cylindrical plastic cups (3 cm diameter × 3.5 cm height) and kept for 24 h to allow for oviposition. Ovipositing females were fed using small pieces of filter paper moistened with 10% honey solution. Cups carrying eggs were kept at the same experimental conditions until hatching by the first instar caterpillars, which were subsequently transferred to large cylindrical plastic containers (11 cm height × 16.5 cm diameter) containing sprouting cowpea grains as a feeding substrate.

Colonies of T. javanus were reared on M. vitrata first instar (three-day-old) caterpillars submitted to parasitization by T. javanus mated females. Parasitized caterpillars were reared on sprouting cowpea grains until pupae stage. Emerged adults were fed with a honey solution.

2.2. Reproductive Tract Morphology and Ovary Anatomy

Three-day-old adult females were dissected in a phosphate-buffered saline (PBS) solution to carefully recover the reproductive system. The specimens were prefixed in 2.5% glutaraldehyde in cacodylate buffer at 4°C during the night. Once fixed, the samples were washed (3 × 10 min) in cacodylate buffer. Postfixation was performed in 2% osmium tetroxide in the same buffer for 1 h at room temperature. Afterwards, the reproductive systems were carefully rinsed with distilled water and washed (3 × 10 min) in 50%, 70%, 90%, and 100% alcohol. Samples were subsequently placed for 1 h in a solution of EMbed 812 Resin (EMS) diluted at 50% in absolute alcohol, were rested overnight at room temperature, and were then transferred to a second, freshly prepared EMbed 812 Resin for 24 h at +60°C for polymerization. Semithin sections were then obtained using an ultra-microtome and stained with methylene blue.

We also examine the egg development within ovariole by dissecting female wasps in PBS at different time intervals after adult emergence (24, 48, 72, and 96 hours). After dissecting the ovaries, ovarioles were removed and fixed in 4% paraformaldehyde. Samples were washed for 5 minutes in PBS and stained either with DAPI (for DNA staining) or phalloidin (for actin staining) by incubation of the specimens for 30 minutes in a solution containing fluorescent phalloidin and DAPI markers diluted at 1/1000 in PBT1%, respectively. Samples were rinsed for 10 minutes in PBS and distilled water and then dried and stored at +4°C for observation of change in the contents of the ovariole using a fluorescence microscope (Zeiss Axiovert 200M equipped with Zeiss AxioCam MRm). Images were processed with ImageJ software .

2.3. Ovariole Counts

To examine the effect of the M. vitrata host quality on ovariole number in adult female, one hundred and sixty (160) each of first instar (two-day-old), old first instar (three-day-old), and second instar (four-day-old) M. vitrata caterpillars, respectively, were submitted individually to parasitization by three-day-old T. javanus females. Caterpillars chosen were well-fed and of uniform size. Each was permitted to be stung once and then reared individually on sprouting cowpea grains in plastic cups (diameter: 9 cm on base and 12 cm on top; height: 4.5 cm) until egression of the parasitoid larva from the host and spinning of the cocoon for the pupal stage. Pupae were then collected in the plastic cups until adult emergence. Thirty-one (31) females (per host group) more than 24 h age were dissected in PBS under a stereomicroscope and the number of ovarioles per ovary was counted.

2.4. Estimation of Egg Production in Female T. javanus

Parasitoid females used in this study were mated and fed using 10% honey solution but not allowed to oviposit. Twenty (20) 12-hour-old and twelve (12) 72-hour-old females were dissected in PBS and observed under a stereomicroscope. Because eggs chambers in T. javanus are translucent white, dissected ovaries were placed in red neutral solution for five (5) minutes to easily observe immature and mature eggs that were thus colored in red. Each ovariole was then detached and opened in PBS solution to count the number of eggs per ovariole. Large size individual eggs, still accompanied by nurse cells, were categorized as “immature eggs” (indicated by solid black color in Figure 1) and well-formed eggs that displayed an ovoid form and had a slender tapering stalk at their posterior end as “mature” eggs (black striped in Figure 1). We counted both immature eggs and mature eggs to estimate the egg production in T. javanus. Small size eggs chambers that were not individually differentiated were not counted (solid white color in Figure 1).

Figure 1: Schematic representation of the ovariole of Therophilus javanus showing differentiated oocyte and accompanying nurse cells (trophocytes) within the ovariole. Immature eggs recorded in our enumeration are large individual egg chambers (follicle) located in the vitellarium whose oocyte displayed an ovoid form and had a slender tapering stalk at their posterior end in solid black color, and mature eggs recorded are large individual egg chambers (egg) of ovoid form that had a slender tapering stalk at their posterior end in black striped color.
2.5. The Effect of Host Age at Oviposition and Female T. javanus Age after Emergence on Mature Egg Production

Maruca vitrata first instar (two-day-old) and second instar (four-day-old) caterpillars, well-fed and of uniform size, were submitted individually for parasitization by three-day-old T. javanus. Each was permitted to be stung once and then reared individually on sprouting cowpea grains in plastic cups (diameter: 9 cm on base and 12 cm on top; height: 4.5 cm) until egression of the parasitoid larva from the host and spinning of the cocoon for the pupal stage. Pupae were then collected in the plastic cups until adult emergence. Thirty (30) emerged female wasps of increasing age (one, two, three, four, and five days after adult emergence) were dissected in PBS under a stereomicroscope. Ovaries were removed and placed in red neutral solution for five minutes. The total number of mature eggs (black striped in Figure 1) in the ovariole was counted per ovary.

2.6. Data Analysis

Data were collected from February 2015 to February 2017 and general linear models with Poisson errors and log-link function, corrected for overdispersion, were used, (1) to test which host caterpillar stages impact the ovariole number in female parasitoid; (2) to test the effect of the female age on (i) the number of eggs (immature eggs + mature eggs) per ovariole, (ii) the number of eggs per ovary, and (iii) the number of eggs per female; (3) to probe the link between the number of ovarioles and the number of eggs in females; and (4) to investigate to what extent host caterpillar stage or parasitoid female age impacts the number of mature eggs per female in T. javanus. Multiple comparisons were carried out using the glht function of the “multcomp” package in the R software [20] to determine significant differences among the mean number of ovarioles per female (at the 0.05 significance level). The statistical software package R 3.3.2 [21] was used for all statistical analyses.

3. Results

3.1. General Morphology of Therophilus javanus Female Reproductive System

The T. javanus female reproductive system consisted of a pair of globular-shaped ovaries housing several ovarioles, a spermatheca, a Dufour’s gland, a venom gland, composed of a venom duct and two venom gland filaments, and the wasp ovipositor (Figure 2).

Figure 2: General morphology of Therophilus javanus female reproductive system, showing the two ovaries (Ov), the venom gland composed by two filaments (Fvg), Dufour’s gland (Dg), the ovipositor (Op), and the two ovipositor sheaths (Ops). Bar 1 mm.
3.2. Impact of Host Quality on the Number of Ovarioles per Female

The mean number of ovarioles per female was found to be significantly influenced by host age at the moment of oviposition (GLM: = 3.6358, , ) (Figure 3). In general, the number of ovarioles varied between the three females categories and was increased as the host caterpillar increased in size at the moment of oviposition. The average count in one-day-old females emerging from L1 two days old, L1 three days old, and L2 four days old is 38.36 ± 4.42 (); 38.16 ± 3.20 (); and 40.87 ± 3.15 (), respectively.

Figure 3: Variation of number of ovarioles in Therophilus javanus female () depending on the host caterpillar age at time of parasitism (two-day-old caterpillars, three-day-old caterpillars, and four-day-old caterpillars).
3.3. Egg Development within the Ovariole

Therophilus javanus ovarioles belong to the polytrophic meroistic type. Egg development occurred anteriorly to posteriorly along the ovariole, with two distinctly recognizable regions: the germarium and the vitellarium. The germarium contained a number of spherical cells observed as either free or clustered (Figure 4(a)). Cell nuclei size increased as they progressed along the germarium. The vitellarium is the posterior region of the ovariole, where egg chambers (follicles) are formed and grown. In T. javanus vitellarium, nurse cells were disposed at the top of the oocyte, all being surrounded by a sheath composed of follicular cells (Figure 4(b)). During progression of the follicles from the anterior to the posterior part of the vitellarium, vitellogenesis takes place and the size of the oocyte increases (Figure 4(c)). A cross-section of the entire ovary shows that egg chambers were in different maturation stages within and between ovarioles (Figure 4(d)). Well-differentiated oocytes (with chorion) displayed an ovoid shaped form and had a slender tapering stalk at their posterior end. Mature eggs measured 160.9 ± 6.9 µm () in length with widths ranging from 25.3 ± 2.6 µm () (anterior pole) to 9.4 ± 1.3 µm () (posterior/basal pole) (Figure 4(e)).

Figure 4: Oogenesis and ovarioles organization in Therophilus javanus. (a) View of the anterior region of T. javanus ovariole (germarium), indicating cell nuclei that increase in size along the germarium. Bar 20 µm. (b) Individual follicle taken out from a T. javanus ovariole. The picture shows the disposition of the nurse cells (Nc) at the top of the oocyte (Oo). The nuclei of follicular cells (Fc) from the sheath surrounding the follicle can also be observed. Bar 50 µm. (c) Basal part of the ovariole of T. javanus. Follicles are in increasing development stages along the vitellarium (from A to B). On the left of the picture, follicles display small oocytes and trophocytes with a large nucleus. On the right, oocytes have increased in size thanks to progression of vitellogenesis. Note the network of actin fibers (in green) surrounding the egg chamber. Nurse cells (Nc); oocyte (Oo). Bar 50 µm. (d) Organization of the ovarioles and follicles within T. javanus ovaries: cross-sections of an ovary containing 15 ovarioles. The ovary is enveloped by the ovarian epithelial sheath (Ov sht) and each ovariole is surrounded by an epithelial sheath (Ovl sht). In the section, oocytes are in different stages within and between ovarioles. Some eggs with a chorion can be observed (shown by arrows). Semithin section stained with methylene blue. Bar 50 µm. (e) The mature egg of T. javanus. The egg has an ovoid shape and a slender tapering stalk at its posterior end. Bar 50 µm.
3.4. Impact of T. javanus Female Age on the Number of Eggs

The number of eggs (both immature and mature) per female ranged from 1 to 88 and from 349 to 476 in 12-hour-old and 72-hour-old females, respectively. The overall mean number per female increased with the female age (GLM: = 6481.2, , ). The number of eggs ranged from 0 to 6 and from 3 to 21 per ovariole, 12 hours and 72 hours after female emergence, respectively (Table 1). As expected, the number of eggs per female was found to be significantly influenced by the total number of ovariole per female (GLM: = 233.4, , ).

Table 1: Egg (immature + mature eggs) number (mean number ± SD) in female Therophilus javanus after emergence. Caterpillars were three days old at the moment of oviposition.
3.5. Impact of Host and Female Wasp Age on the Egg Load

Overall, the mean number of mature eggs per female was found to be significantly influenced by host age (GLM: = 44.4, df = 1, ) and parasitoid female age (GLM: = 16600.9, df = 4, ). Females that emerged from four-day-old host caterpillar at oviposition had a higher mean number of mature eggs. (Figure 5).

Figure 5: Effect of host age (at oviposition) and female age on the mature egg (individual egg chambers located in the vitellarium who displayed an ovoid form and had a slender tapering stalk at their posterior end (in black striped color in Figure 1)) load in Therophilus javanus. Caterpillars were two and four days old at the moment of oviposition. Error bars represent the standard errors of the means (). Means that were significantly different between two- and four-day-old hosts only according to GLM and Tukey HSD test are indicated by asterisks (; ; ns: nonsignificant).

4. Discussion

Therophilus javanus belongs to an important braconid family, the Agathidinae, whose reproductive biology is largely unknown. Our study is the first of its kind highlighting major characteristics of T. javanus reproductive biology and provides the basis for deploying this parasitoid as a biological control agent against the cowpea pod borer M. vitrata in West Africa and elsewhere.

The female reproductive tract of T. javanus presents a classical basic morphological organization, similar to the one described in braconids and other Agathidinae species, e.g., Agathis pumila (Hymenoptera: Braconidae) (Ratzeburg) [10]. However, differently from other braconids, the follicles are not organized in a string within T. javanus ovarioles but rather appear as “free” egg chambers. This kind of organization resembles to some extent what have been described in some Eulophidae parasitoids (e.g., Palmistichus elaeisis (Hymenoptera: Eulophidae)) [22].

In contrast to the insect model Drosophila, scarce data is available on oocyte size and number and on ovariole number in parasitoid species [23]. In some parasitoid species (e.g., ichneumonids), the number of ovarioles was shown to be a good indicator of fecundity [24]. Ovariole number is largely species-dependent but may show plasticity as a function of biological or environmental factors [2527]. Our observations suggest that T. javanus displays quite a variable number of ovarioles, which has also been commonly reported in noncyclostome Braconidae subfamilies, including in Agathidinae (from 4 to 30) [6, 28]. In T. javanus, the highest number of ovarioles has been observed in females issued from larger hosts (i.e., second instar M. vitrata larvae). Impact of host instar on parasitoids ecological and biological traits has been reported in several studies [2932]. For example, the average number of eggs in Microplitis rufiventris Kok (Hymenoptera: Braconidae) was higher in females that emerged from Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) parasitized at younger larval stage [33].

Our study has demonstrated that, in T. javanus, egg load is influenced by females’ age. The largest average number of mature eggs (177.97 ± 2.62) was counted in five-day-old females that emerged from larger hosts (L2, four-day-old). This confirms observations in other Braconidae that larger parasitoids are issued from larger host larvae and larger females lay higher number of eggs [34]. In spite of the uniform size of M. vitrata caterpillars used for parasitization, we did observe a variable number of eggs in T. javanus females, which has been demonstrated to depend on abiotic or biotic factors in the Agathidinae B. vulgaris and A. gibbosa [9, 11]. For instance, the number of eggs laid by B. vulgaris varies depending on the size of the wasp female and on temperature at adult emergence, whereas mating is known to decrease egg load in A. gibbosa. In three-day-old mated T. javanus females, the mean number of mature eggs was much smaller than the number of immature ones (50 and 357, resp.), suggesting a large potential fecundity. As generally observed in koinobiont species including some Agathidinae [35], this could be related to the ability of T. javanus females to continue oogenesis after emergence, provided there are sufficient protein sources to maintain oogenesis and complete egg maturation. In fact, the impact of adult life time protein deficiency on oogenesis has been previously documented in Microterys flavus (Hymenoptera: Encyrtidae) [36]. Like for A. pumila [10], T. javanus females do not host-feed and may probably need to take additional protein from nonhost food sources after emergence, possibly slowing down egg maturation.

As described for other koinobiont species that attack hosts at early stages [35], T. javanus displays very tiny eggs (0.1 mm in length and 0.025 mm for its larger width). This minute size and the tear shape of T. javanus eggs correspond to previous descriptions of Agathidinae eggs such as those produced by B. vulgaris (Cress.), A. pumila, and A. gibbosa [911]. T. javanus females are synovigenic; that is, they emerge with high numbers of immature eggs and only few mature eggs but continue to produce eggs throughout the adult stage, implying that females can start to oviposit in host caterpillars just after emergence. This ability to start oviposition just after emergence has been mentioned in other Agathidinae species, that is, A. gibbosa, A. pumila, and B. vulgaris [911]. In contrast to what was observed in the ovarioles of A. pumila and most Braconidae, however, both developing and mature eggs were found at the same level in the basal part of T. javanus ovariole. This suggests that T. javanus females might have developed a mechanism allowing them to lay only mature eggs in the host.

The current mass-rearing protocol, using three-day-old T. javanus females, stems from a desire to maximize the production of mated females as recommended for parasitoid rearing in biological control programs [37, 38]. Our findings, however, show that egg production was relatively low (40.8 ± 8.6) during the first three days following adult emergence, suggesting that better outputs could be obtained using females older than three days. Our results also show that egg production in T. javanus is influenced by the size or instar of the caterpillar host. From a mass-rearing perspective, this suggests that the overall fecundity of T. javanus could be improved by selecting second instar caterpillars. However, under field conditions, the fecundity of foraging T. javanus females could be influenced by the size of the available M. vitrata life stages. Also, along with preliminary observations that T. javanus females did not perform host feeding on M. vitrata caterpillars, it will be important to investigate how feeding on sugar sources may impact the fecundity of T. javanus females. In fact, synovigenic parasitoids that do not feed on host are usually able to use sugar foods for oogenesis [18]. Notably, cowpea itself, the major crop hosting caterpillars of M. vitrata, secretes extrafloral nectar [39], which may provide an adequate source of sugar food for foraging female parasitoids. We expect similar extrafloral nectar to be present on other important, wild-occurring host plants such as Sesbania rostrata (Fabales: Fabaceae) and Tephrosia platycarpa (Fabales: Fabaceae), known to harbor important pod borer populations [40], which might also be visited by foraging T. javanus females.

5. Conclusion

Biological, rather than pesticidal, control of M. vitrata offers numerous advantages, especially in poor rural areas where the cost of pesticides, along with human and environmental exposures, becomes unsustainable or prohibitive. Even with pesticides, however, difficulty in recognizing the presence of M. vitrata prior to destructive crop predation makes conventional crop protection methods challenging. Our findings provide the first baseline information toward elucidating several factors influencing the reproductive biology in T. javanus, a promising biological control candidate against M. vitrata in West Africa. The fact that T. javanus, along with its high level of potential fecundity, may also demonstrate a greater facility for identifying and taking advantage of the presence of M. vitrata potentially enhances its use as a biological control to a great degree, while also affording the many cost, human, and environmental advantages of not using chemical pesticides.

Conflicts of Interest

All authors declare no conflicts of interest.


The authors are grateful to the Cooperation and Cultural Action Service (SCAC) of the French Embassy in Cotonou for partial support of DAS (no. 836329C and no. 861327C) and to the Bill and Melinda Gates Foundation (BMGF) for partial cofunding of the study and open access publication support. They thank Dr. Srinivasan Ramasamy of The World Vegetable Center (AVRDC), Taiwan, for providing the rearing colony of T. javanus used for their studies. The authors thank Mathias Azokpota, Véronique Jouan, and Benjamin Datinon for their technical assistance.


  1. S. R. Singh, L. E. N. Jackai, J. H. R. Dos Santos, and C. B. Adalla, “Insect Pests of Cowpea,” in Insect Pests of Tropical Food Legumes, Singh. S. R., Ed., pp. 43–90, J. Wiley & Sons Ltd, Chichester, England, UK, 1990. View at Google Scholar
  2. M. Periasamy, R. Schafleitner, K. Muthukalingan, and S. Ramasamy, “Phylogeographical structure in mitochondrial DNA of legume pod borer (Maruca vitrata) population in tropical Asia and sub-Saharan Africa,” PLoS ONE, vol. 10, no. 4, Article ID e0124057, 2015. View at Publisher · View at Google Scholar · View at Scopus
  3. D. T. Dung, L. Phuong, and K. D. Long, “Insect parasitoid composition on soybean, some eco-biological characteristics of the parasitoid, Xanthopimpla punctata (Fabricius) on soybean leaf folder Omiodes indicata (Fabricius) in Hanoi, Vietnam,” Journal of International Society for Southeast Asian Agricultural Sciences, vol. 17, pp. 58–69, 2011. View at Google Scholar
  4. R. Srinivasan, S. Yule, J. Chang et al., “Towards developing a sustainable management strategy for legume pod borer, maruca vitrata on yard-long bean in Southeast Asia,” in Proceedings of the Regional Symposium on High Value Vegetables in Southeast Asia: Production, Supply and Demand, R. Holmer, G. Linwattana, P. Nath, and J. Keatinge, Eds., AVRDC; The World Vegetable Center, Chiang Mai, Thailand, 2012. View at Publisher · View at Google Scholar
  5. M. J. Sharkey, “A taxonomic revision of Alabagrus (hymenoptera: braconidae) , bulletin of the British museum (natural history),” Entomology, vol. 57, no. 2, pp. 311–437, 1988. View at Google Scholar
  6. D. L. J. Quicke, The Braconid and Ichneumonid Parasitoid Wasps, Biology, Systematics , Evolution and Ecology, John Wiley & Sons, Hoboken, NJ, USA, 2015.
  7. M. J. Sharkey, “Subfamily Agathidinae,” in Proceedings of the Manual of the New World genera of the family Braconidae (Hymenoptera) Special Publication of the International Society of Hymenopterists 1, R. A. Wharton, P. M. Marsh, and M. J. Sharkey, Eds., pp. 69–83, International Society of Hymenopterists, Washington, DC, 1997.
  8. D. H. Janzen, M. J. Sharkey, and J. M. Burns, “Parasitization biology of a new species of Braconidae (Hymenoptera) feeding on larvae of Costa Rican dry forest skippers,” Tropical Lepidoptera, vol. 9, no. 2, pp. 33–41, 1998. View at Google Scholar
  9. J. A. Odebiyi and E. R. Oatman, “Biology of agathis gibbosa (Hymenoptera: braconidae), a primary parasite of the potato tuberworm,” Annals of the Entomological Society of America, vol. 65, no. 5, pp. 1104–1114, 1972. View at Publisher · View at Google Scholar
  10. F. W. Quednau, “Notes on life-history, fecundity, longevity, and attack pattern of agathls pumila (hymenoptera: braconidae), a parasite of the larch casebearer,” The Canadian Entomologist, vol. 102, no. 6, pp. 736–745, 1970. View at Publisher · View at Google Scholar · View at Scopus
  11. F. J. Simmonds, “The biology of the parasites of loxostege sticticalis, L., in North America—bracon vulgaris (cress.) (braconidae, agathinae),” Bulletin of Entomological Research, vol. 38, no. 1, pp. 145–155, 1947. View at Publisher · View at Google Scholar · View at Scopus
  12. M. R. Shaw and T. Huddleston, “Classification and biology of braconid wasps,” in Handbooks for the Identification of British Insects, Royal Entomological Society of London, London, UK, 1991. View at Google Scholar
  13. B. D. Roitberg, G. Boivin, and L. E. M. Vet, “Fitness, parasitoids, and biological control: an opinion,” The Canadian Entomologist, vol. 133, no. 3, pp. 429–438, 2001. View at Publisher · View at Google Scholar · View at Scopus
  14. J. David, “Le nombre d'ovarioles chez drosophila melanogaster: relation avec la fecondite et valeur adaptative,” in Archives de zoologie expérimentale et générale, vol. 111, pp. 357–370, 1970. View at Google Scholar
  15. J. Büning, The Insect Ovary: Ultrastructure, Previtellogenic Growth And Evolution, Chapman & Hall, London, UK, 1994.
  16. M. P. Kambysellis and W. B. Heed, “Studies of oogenesis in natural populations of drosophilidae. i. relation of ovarian development and ecological habitats of the hawaiian species,” The American Naturalist, vol. 105, no. 941, pp. 31–49, 1971. View at Publisher · View at Google Scholar
  17. S. E. Flanders, “Regulation of ovulation and egg disposal in the parasitic hymenoptera,” The Canadian Entomologist, vol. 82, no. 6, pp. 134–140, 1950. View at Publisher · View at Google Scholar · View at Scopus
  18. M. A. Jervis, J. Ellers, and J. A. Harvey, “Resource acquisition, allocation, and utilization in parasitoid reproductive strategies,” Annual Review of Entomology, vol. 53, pp. 361–385, 2008. View at Publisher · View at Google Scholar · View at Scopus
  19. A. Rivero, D. Giron, and J. Casas, “Lifetime allocation of juvenile and adult nutritional resources to egg production in a holometabolous insect,” Proceedings of the Royal Society B: Biological Sciences, vol. 268, no. 1473, pp. 1231–1237, 2001. View at Publisher · View at Google Scholar · View at Scopus
  20. F. Bretz, T. Hothorn, and P. Westfall, Multiple Comparisons Using R. Chapman and Hall: CRC, Chapman and Hall/CRC, 2010. View at Publisher · View at Google Scholar
  21. R Core Team, “R: A language and environment for statistical computing,” Retrieved from,, 2016.
  22. G. S. Andrade, A. H. Sousa, J. C. Santos, F. C. Gama, J. E. Serrão, and J. C. Zanuncio, “Oogenesis pattern and type of ovariole of the parasitoid Palmistichus elaeisis (Hymenoptera: Eulophidae),” Anais da Academia Brasileira de Ciencias, vol. 84, no. 3, pp. 767–774, 2012. View at Publisher · View at Google Scholar · View at Scopus
  23. M. A. Jervis, M. Copland, and J. A. Harvey, “The life-cycle,” in Insects as Natural Enemies: A Practical Perspective, M. A. Jervis, Ed., pp. 73–165, Springer, Dordrecht, Netherlands, 2007. View at Google Scholar
  24. P. W. Price, Evolutionary Strategies of Parasitic Insects and Mites, Springer US, Boston, MA, 1975. View at Publisher · View at Google Scholar
  25. J. Büning, “The ovariole: structure, type, and phylogeny,” in Microscopic anatomy of invertebrates 11C Insecta, F. Harrison and M. Locke, Eds., pp. 897–932, Wiley-Liss, New York, 1998. View at Google Scholar
  26. T. A. Markow and P. M. O'Grady, “Drosophila biology in the genomic age,” Genetics, vol. 177, no. 3, pp. 1269–1276, 2007. View at Publisher · View at Google Scholar · View at Scopus
  27. M. Telonis-Scott, L. M. McIntyre, and M. L. Wayne, “Genetic architecture of two fitness-related traits in drosophila melanogaster: ovariole number and thorax length,” Genetica, vol. 125, no. 2-3, pp. 211–222, 2005. View at Publisher · View at Google Scholar · View at Scopus
  28. K. Iwata, “The comparative anatomy of the ovary in Hymenoptera, Part III. Braconidae (including Aphidiidae) with descriptions of ovarian eggs,” Kontyû, vol. 27, pp. 231–238, 1959. View at Google Scholar
  29. J. A. Harvey, I. F. Harvey, and D. J. Thompson, “Flexible larval growth allows use of a range of host sizes by a parasitoid wasp,” Ecology, vol. 75, no. 5, pp. 1420–1428, 1994. View at Publisher · View at Google Scholar · View at Scopus
  30. J. A. Harvey and L. E. M. Vet, “Venturia canescens parasitizing galleria mellonella and anagasta kuehniella: differing suitability of two hosts with highly variable growth potential,” Entomologia Experimentalis et Applicata, vol. 84, no. 1, pp. 93–100, 1997. View at Publisher · View at Google Scholar · View at Scopus
  31. C. Ozkan, “Effect of food, light and host instar on the egg load of the synovigenic endoparasitoid venturia canescens (hymenoptera: ichneumonidae),” Journal of Pest Science, vol. 80, no. 2, pp. 79–83, 2007. View at Publisher · View at Google Scholar · View at Scopus
  32. R. Sequeira and M. Mackauer, “Nutritional ecology of an insect host-parasitoid association: the pea aphid-Aphidius ervi system,” Ecology, vol. 73, no. 1, pp. 183–189, 1992. View at Publisher · View at Google Scholar · View at Scopus
  33. W. E. Khafagi, E. M. Hegazi, P. Andersson, and F. Schlyter, “Does host size and feeding status influence the egg load of microplitis rufiventris (Hymenoptera: Braconidae)?” Annals of the Entomological Society of America, vol. 104, no. 2, pp. 221–228, 2011. View at Publisher · View at Google Scholar · View at Scopus
  34. P. G. Tillman and J. R. Cate, “Effect of host size on adult size and sex ratio of Bracon mellitor (Hymenoptera, Braconidae),” Environmental Entomology, vol. 22, no. 5, pp. 1161–1165, 1993. View at Publisher · View at Google Scholar · View at Scopus
  35. M. R. Strand and J. Casas, “parasitoid and host nutritional physiology in behavioral,” in Behavioral Ecology of Parasitoids: from Theoretical Approaches to Field Applications, E. Wajnberg, J. Van Alphen, and C. Berstein, Eds., pp. 113–128, Blackwell Publishing, London, UK, 2008. View at Google Scholar
  36. B. R. Bartlett, “Patterns in the host-feeding habit of adult parasitic hymenoptera,” Annals of the Entomological Society of America, vol. 57, no. 3, pp. 344–350, 1964. View at Publisher · View at Google Scholar
  37. I. Hardy, P. Ode, and M. Siva-Jothy, “Mating behavior,” in Insects as Natural Enemies: A Practical Perspective, M. A. Jervis, Ed., pp. 219–260, Springer, Dordrecht, Netherlands, 2005. View at Google Scholar
  38. A. L. Joyce, R. E. Hunt, J. S. Bernal, and S. Bradleigh Vinson, “Substrate influences mating success and transmission of courtship vibrations for the parasitoid Cotesia marginiventris,” Entomologia Experimentalis et Applicata, vol. 127, no. 1, pp. 39–47, 2008. View at Publisher · View at Google Scholar · View at Scopus
  39. J. S. Pate, M. B. Peoples, P. J. Storer, and C. A. Atkins, “The extrafloral nectaries of cowpea (Vigna unguiculata (L.) Walp.) II. Nectar composition, origin of nectar solutes, and nectary functioning,” Planta, vol. 166, no. 1, pp. 28–38, 1985. View at Publisher · View at Google Scholar · View at Scopus
  40. D. Y. Arodokoun, M. Tamò, C. Cloutier, and R. Adeoti, “Importance of alternative host plants for the annual cycle of the legume pod borer, Maruca vitrata fabricius (Lepidoptera: Pyralidae) in southern and central Benin,” International Journal of Tropical Insect Science, vol. 23, no. 2, pp. 103–113, 2003. View at Google Scholar · View at Scopus