Research Article |
Corresponding author: Cleder Pezzini ( cleder.pezzini@hotmail.com ) Academic editor: Jose Fernandez-Triana
© 2017 Cleder Pezzini, Simone Mundstock Jahnke, Andreas Köhler.
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:
Pezzini C, Jahnke SM, Köhler A (2017) Morphological characterization of immature stages of Habrobracon hebetor (Hymenoptera, Braconidae) ectoparasitoid of Ephestia kuehniella (Lepidoptera, Pyralidae). Journal of Hymenoptera Research 60: 157-171. https://doi.org/10.3897/jhr.60.20104
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Habrobracon hebetor (Say) is a cosmopolitan idiobiont braconid which parasitizes Pyralidae larvae, a pest of stored products, such as Ephestia kuehniella (Zeller). The objective was to describe the morphology of immature forms of H. hebetor and morphological changes throughout its development. Mated females of H. hebetor were individualized in Petri dishes containing larvae of E. kuehniella for parasitism for six hours. Then, the females were removed, leaving only the eggs placed on the host. The development was evaluated every 12 hours, recording all stages and changes until the emergence of adults. Using stereoscopic optical and scanning electron microscopy, photographs of immature individuals were taken. The results showed that this parasitoid completes its development between 10–12 days. There were stages overlaps during egg to adult development. Eggs are hymenopteriform, with a smooth surface. According to cephalic capsule and larval length measurements of H. hebetor, it was possible to determine four instars. In general, the instars are similar to each other, differing mainly in the size and shape of segments. Larvae present a gradual loss of transparency, becoming opaquer at each successive instar. Last instar larvae distanced from the host to form the cocoon and to pupate. This study was relevant for a better understanding of the physiological interactions between E. kuehniella and H. hebetor.
Bionomy, Pre-imaginal period, Parasitoid, Electro micrographs
In Hymenoptera 15 superfamilies and 62 families of parasitoid are recognized (
Habrobracon hebetor (Say) (Hymenoptera: Braconidae) is an idiobiont ectoparasitoid with a cosmopolitan distribution (
The number of instars, development time and feed consumption of H. hebetor was evaluated by
The morphological characteristics of immature stages play an important role in the recognition, taxonomy and classification of parasitoid wasps (
In taxonomic studies, most morphological descriptions of Braconidae are concentrated in the adult stage, while the biology and morphology of immature stages still lack information (
Although some aspects of biology at immature stages of H. hebetor have been studied, researches are mainly focused on the interactions between parasitoid, host and environmental factors (
Rearings of H. hebetor and its host E. kuehniella were kept in the Laboratory of Entomology of the University of Santa Cruz do Sul (
Twenty mated females of H. hebetor were individualized in Petri dishes containing one larva of E. kuehniella for parasitism for six hours. Then, the females were removed and leaving just one egg placed on the host, the others removed. The development time was evaluated every 12 hours by recording all stages and changes until the emergence of adults.
For the determination of the number of larval instars, a segmented regression model was used according to
A sample of each stage was fixed in 25% glutaraldehyde solution in a 0.2 M phosphate buffer and distilled water for 14 days. The samples were then washed three times (30 min/wash) in 0.2 M phosphate and distilled water (1:1 ratio) dehydrated in a graduated series of acetone (30, 50, 70, 90 and 100%). They were dried (Balzers CPD030) and metallized (Balzers SCD050), after which a scanning electron microscope (Jeol JSM 6060) was performed. Electro micrographs of all immature stages were made.
To the description of each stage, one individual at every stage with an average age was used. Comments on morphological changes that may occur during each stage were added after the diagnoses. For cephalic capsule chaetotaxy, last instar larvae were used as model since the distribution of setae is the same for all larval instars and prepupa (Short, 1952).
Morphological terminologies (Fig.
Abbreviations:
A abdominal segment
AS anal segment
T thorax segment
The examined material was registered at the Entomological Collection of Santa Cruz do Sul (CESC), under lots no. 79,495 (Egg), 79,496 (Larva, 1st instar), 79,497 (Larva, 2nd instar), 79,498 (Larva, 3rd instar), 79,499 (Larva, 4th instar), 79,500 (Prepupa), 79,501 (female pupa) and 79,502 (male pupa).
There was stage overlap (between replicates) during the egg to adult development of H. hebetor, indicating variations in development time. The amplitude of each immature stage was small, especially in the first instars. Thus, four days after oviposition, the larvae were already at the fourth and last instar, initiating the formation of the cocoon to pupate. The pupal phase was the longest of all development stages, lasting more than four days (Table
The diagnosis of the entire development of H. hebetor is presented below with photographs and electro micrographs presenting some details of each stage, distribution and nomenclature of setae on the cephalic capsule, and comments on the morphological changes at each stage.
Diagnosis: opaque white, with a smooth surface (Fig.
Measurements: overall length: 0.52 mm; maximum width: 0.12 mm.
Comments: approximately 12 hours after oviposition, it is possible to observe the embryo in formation and its development (Fig.
It was possible to determine four larval instars of H. hebetor based on cephalic capsule length and larval body length, (Table
In general, the four larval instars are similar to each other, differing mainly in the size and shape of segments. Larvae present a gradual loss of transparency, becoming opaquer at each successive instar with the enlargement of the intestine. Each instar had a different development time (Table
Development time and sizes of immature stages of Habrobracon hebetor in Ephestia kuehniella larvae (12 h photophase, 28 ± 2°C and 50 ± 20% RH).
Stage/instar | Duration (Days ± SD) | Body length (mm ± SD) | Maximum body width (mm ± SD) | Cephalic capsule width (mm ± SD) |
Egg | 1.35 ± 0.343 | 0.52 ± 0.056 | 0.12 ± 0.011 | - |
Larva 1 | 0.73 ± 0.089 | 0.44 ± 0.073 | 0.10 ± 0.019 | 0.10 ± 0.014 |
Larva 2 | 0.41 ± 0.050 | 0.89 ± 0.142 | 0.36 ± 0.022 | 0.18 ± 0.019 |
Larva 3 | 0.90 ± 0.110 | 1.87 ± 0.283 | 0.60 ± 0.086 | 0.24 ± 0.019 |
Larva 4 | 1.22 ± 0.150 | 2.67 ± 0.139 | 0.90 ± 0.079 | 0.30 ± 0.026 |
Prepupa | 1.97 ± 0.374 | 2.90 ± 0.182 | 0.84 ± 0.035 | 0.38 ± 0.022 |
Female pupa | 4.47 ± 0.413 | 2.46 ± 0.113 | 0.91 ± 0.037 | 0.56 ± 0.011 |
Male pupa | 2.50 ± 0.164 | 0.78 ± 0.027 | 0.56 ± 0.013 |
Diagnosis: spherical cephalic capsule, width equal to the length of three thorax segments together, visible short antennae below the vertex region, sparse setae in the frontal region of the cephalic capsule (Fig.
Measurements: overall length: 0.42 mm; cephalic capsule: 0.10 mm; maximum width: 0.10 mm.
Comments: at the initial phase of the first instar, the larva is translucent and the cephalic capsule is as wide as the following segments. As the larva develops, its body grows rapidly and the segmentation becomes more noticeable. During the first instar, the thoracic segments exceeded the width of the cephalic capsule.
Diagnosis: spherical cephalic capsule, almost twice as wide as that of the first instar, short visible antennae with sparse setae in the frontal region of the cephalic capsule (Fig.
Measurements: overall length: 0.92 mm; cephalic capsule: 0.18 mm; maximum width: 0.36 mm.
Comments: the width of the cephalic capsule increases, but less than the width of the body segments. With the increase in larval body size, the intestine occupies an increasing volume, reaching up to a third of the body’s space at this phase of development.
Diagnosis: spherical cephalic capsule 2.5 times larger than that of the first instar, visible short antennas with sparse setae in the frontal region of the cephalic capsule (Fig.
Measurements: overall length: 1.52 mm; cephalic capsule: 0.24 mm; maximum width: 0.62 mm.
Comments: as the body segments increase in size, the cephalic capsule begins to be covered in the back by the T1, reaching up to a third of the cephalic capsule. Unlike the first and second instar, the surface has short, dense setae, easily visible, scattered across the thorax and abdomen. Trichoid sensilla are developed, with a base and cone shape, reaching twice the size of setae. The larva becomes opaquer and the intestine occupies two-thirds of the body at this instar.
Diagnosis: spherical cephalic capsule 3 times larger than that of the first instar, visible short antennas with sparse setae in the frontal region of the cephalic capsule (Fig.
Measurements: overall length: 2.64 mm; cephalic capsule: 0.29 mm; maximum width: 0.95 mm.
Comments: as they grow, body segments increase in size, making the cephalic capsule proportionally the smallest part of the larva in addition to being almost totally involved dorsally by the first thoracic segment. Many granules appear as small white patches scattered under the cuticle of the abdominal segments. Approximately 84 h after parasitism, larvae of the fourth instar had already consumed almost all the tissues of the host and distanced themselves from it to initiate the formation of the cocoon, which is woven with silk produced by the labial glands, forming a thick layer of threads over its body (Fig.
Diagnosis: cephalic capsule distinctly separated from the rest of the body, with an enlargement of the posterior lobe (Fig.
Measurements: overall length: 2.90 mm; cephalic capsule: 0.38 mm; maximum width: 0.84 mm.
Comments: at the end of the fourth instar, the prepupal transformation occurs with the fully formed, oval-shaped and white-shaped cocoon, a lighter color and absence of movement. With the connection of the midgut to the posterior intestine, there is the elimination of the meconium, which is adhered to the cocoon, making the intestine translucent. There is a differentiation in the cephalic capsule with the expansion of the posterior lobe, where, at the end of this stage, the composite eyes and the three dorsal ocelli become visible, presenting a reddish-brown coloration.
Female diagnosis: characteristics of the head as in adults, pigmented eyes and ocelli fully formed, antennas curved down to the thorax, ending in the insertion of the last pair of legs, 13 flagellomeres of equal size, 1.5 times wider than long, containing a ring of eight spines in each segment, spines at the base as large as long with approximately half the length of each flagellomere, buccal apparatus with sclerotized mandibles, thorax as in adults but with wing structures folded laterally to the thorax, reaching A2, legs developed close to the body, abdomen with nine segments, ovipositor curved upward at the back of the abdomen (Fig.
Measurements: overall length: 2.46 mm; cephalic capsule: 0.56 mm; maximum width (abdomen): 0.94 mm.
Male diagnosis: pupa similar to that of female, differing in the longest antennas reaching ventrally A5, containing 20 equal-sized flagellomeres, as large as long, with a ring of eight spines in each segment, spines at the base as large as long with approximately one-third of the length of each flagellomere, absent ovipositor (Fig.
Measurements: overall length: 2.50 mm; cephalic capsule: 0.56 mm; maximum width (abdomen): 0.76 mm.
Comments: exarate pupa is protected by the cocoon produced by the last instar larva. Initially, only eyes and ocelli are pigmented (Fig.
Scanning electron micrographs of immature stages of Habrobracon hebetor: A detail of the smooth surface of the egg B detail of the setae on the dorsal surface of the thorax and abdomen of third and fourth larval instars and prepupa C detail of the smooth dorsal surface of first and second larval instars with spiracles D detail of a trichoid sensillum E prepupa F female pupa G male pupa.
The time of development from egg to adult emergence was similar to that reported for H. hebetor by
This work evaluates in more depth information about the development stages of H. hebetor reported in the article by
The detailed diagnoses of all immature stages are a complement to the study conducted by
Eggs with an elongated hymenopteriform shape were expected because, as
The existence of four larval instars of H. hebetor was found by adjusting the total length and width of the cephalic capsule measurements data by a segmented regression model (
According to
Larvae with low structural complexity and successive pigmentation changes in body color had been reported for other species of parasitoids.
In the third and fourth larval instars were observed short and dense setae on all dorsal surface of the thorax and abdomen. In the description of B. brevicornis made by
The appearance of white granules along the abdomen at the last larval instar has also been reported for a parasitoid of the same superfamily, Diadromus collaris (Gravenhorst) (Hymenoptera: Ichneumonidae) (
Four days after oviposition, the parasitoid is already at the last larval instar and moves away from the host. This behavior is justified because, as reported by
After leaving the host, the parasitoid begins the construction of the cocoon to pupate. The formation of the cocoon with a thick layer of silk, according to
The development of H. hebetor is similar in many ways to other braconids. However, in this study, we documented the whole development of H. hebetor, including morphological changes, thus providing a detailed basis for the morphological characterization of the immature stages and the development of H. hebetor.
To the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the master scholarship granted to the first author. To MCTI/CT-Agro/CNPq 38/2013 and Japan Tobacco International (JTI) for the financial support. To the Centro de Microscopia e Microanálise of UFRGS for the technical support to perform the electromicrographs. To Dr. Alexandre Somavilla, from INPA, for the review of the work.