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Bioassays to Investigate the Effects of Insect Oviposition on a Plant’s Resistance to Herbivores
生物测定法研究昆虫产卵对植物抵御食草动物的影响   

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Abstract

Plants respond to herbivory with diverse defence responses (Schoonhoven et al., 2005). Many herbivorous insects deposit their eggs on their host plants before their larvae start to feed. Thus, plants could use insect eggs as a signal to increase their resistance to herbivores. Here, we report experimental procedures to explore whether and how insect oviposition impacts on plant resistance against the feeding larvae. The described approach revealed that Nicotiana attenuata (N. attenuata) plants that were previously exposed to oviposition by lepidopteran moths respond to herbivory by generalist Spodoptera exigua (S. exigua) and specialist Manduca sexta (M. sexta) larvae with an increased induction of defence responses, which results in a decreased performance or immune state of the feeding larvae (Bandoly et al., 2015; Bandoly et al., 2016). Consequently, insect oviposition can prime feeding-induced plant defence (priming: an enhanced plant response to stress upon the experience of a prior stimulus; Hilker et al., 2015). Full-factorial experiments with standardised procedures for insect oviposition and larval herbivory allow to decipher the effect of the plant exposure to insect eggs on the larval performance, feeding damage and immune state as well as to discriminate egg-induced plant responses from egg-primed responses to larval feeding.

Materials and Reagents

  1. Reaction tubes, 0.2 ml (e.g., Carl Roth GmbH + Co. KG, catalog number: H560.1 )
  2. Reaction tubes, 1.5 ml or 2 ml (e.g., Carl Roth GmbH + Co. KG, catalog number: CK06.1 )
  3. Nicotiana attenuata Torr. ex. Watson (Solanaceae), 4-5 weeks old (rosette stage)
  4. Spodoptera exigua Hübner (Noctuidae, Lepidoptera) or Manduca sexta Linnaeus (Sphingidae, Lepidoptera)
  5. Liquid nitrogen
    Note: All materials and equipment used is rather general lab equipment or house ware and we just referred to example products we used.

Equipment

  1. General equipment
    1. A soft paint brush [e.g., Rotmarder 122A, size 2 (Habico, catalog number: 50122A10 )]
    2. Featherweight forceps (e.g., Carl Roth GmbH + Co. KG, catalog number: AN00.1 )
    3. Rearing boxes (e.g., 14 x 21 x 5 cm), lid with gauze (nylon mesh 0.12 mm width)
    4. Precision balance (e.g., Sartorius AG, model: Sartorius MC210S )

  2. Oviposition on plants
    1. Flight cages [e.g., kweekkooi, 60 x 60 x 90 cm (Vermandel, catalog number: 80.304 ) or flexarium, 42 x 42 x 76 cm (Rolf C. Hagen Inc., Exo Terra, model: PT2552 )]
      Note: Flight cages eventually with slots at the cage sides through which defined leaves can be exposed and that can be closed with small claw hair clips.
    2. Hanging labels [e.g., Hängeetiketten, HERMA FACHSHOP, model: 6901 ]] or a thread
    3. Headlamp (e.g., Petzl, model: PIXA® 3 )

  3. Larval performance, feeding damage and haemolymph sampling
    1. Vented clip cages [e.g., made of two dressing cups (e.g., Ø 7.03, 2.3 cm (KIV-KREIS, catalog number: 770405509 )) with polyurethane foam at the rims, the bottom of one cup is replaced by gauze (nylon mesh 0.12 mm width) and the cage can be closed with small claw hair clips]
    2. Folding magnifier [e.g., TRIPLET 20x, 21 mm (Light In The Box Ltd., catalog number: 01239576 )]
    3. Laboratory support stand with clamps
    4. White board with reference areas (e.g., a laminated sheet of white paper with black squares of 1 x 1 cm)
    5. Camera [e.g., Canon EOS 1200D (Canon Inc., model: Canon EOS 1200D ) with EFS 18-25 mm macro lenses (Canon Inc., model: Canon EF-S 18-55mm f/3.5-5.6 IS II)]
  4. Standardised damage and harvest of leaf tissue for analyses of plant defence traits
    1. Vacuum pump (e.g., Vacuubrand, model: PC 510 )
    2. Teflon tube [e.g., outer Ø 1.6 mm (VWR International, catalog number: BOHLS1810-01 )]
    3. Glass vial with rubber septum cap [e.g., 4 ml HPLC vial (Techlab, catalog number: FLK 101.243 and FLK 101.972 )]
    4. Tracing/Pattern wheel (e.g., Nähmit, catalog number: 070210 )
    5. 10 µl pipette
    6. Scalpel (e.g., Carl Roth GmbH + Co. KG, catalog number: X004.1 )
    7. Dissecting forceps (e.g., Carl Roth GmbH + Co. KG, catalog number: 2690.1 )

Procedure

  1. Oviposition on plants
    1. Sort S. exigua or M. sexta pupae for their sex. Figure 1 depicts the discrimination characteristics between male and female lepidopteran pupae.
    2. Let the pupae eclose in the flight cages used for oviposition. In case of S. exigua place 15 female and 15 male pupae in a flight cage. In case of M. sexta place 8 female and 8 male pupae in a flight cage. Inspect the cages twice per day (in the morning and in the evening) for eclosed moths. To ensure that the moths had time to mate and have started to lay eggs use the cages 2 d after eclosion for oviposition in case of S. exigua and 3 days after eclosion in case of M. sexta.


      Figure 1. Abdomen of male (A) and female (B) Spodoptera exigua pupae and of male (C) and female (D) Manduca sexta pupae. Arrows indicate genital structures at the ventral side that differ between sexes in morphology and the segments they are located. Photographs: Stefanie Luka (A, B), Anke Steppuhn (C, D). Scale bars, 2 mm (A, B), 5 mm (C, D)

    3. To equalise slight differences in plant ontogeny between treatment groups, sort 4-5 weeks old Nicotiana attenuata rosette stage plants [grown as described in Krügel et al. (2002); Figure 2A] by size (i.e. rosette diameter) and elongation state (degree of stalk development) and assign equal plants to all treatments of each biological replicate. For a full-factorial experimental design that enables to evaluate effects of oviposition on plant defence parameter four treatment groups are required (Box 1).

      Box1. Treatments required for a full-factorial priming experiment
      I. both oviposition and larval feeding after the natural egg incubation time
      II. oviposition not followed by larval feeding
      III. larval feeding without prior oviposition
      IV. control plants without oviposition and larval feeding

    4. To control for the experimental procedure and other stimuli associated with exposure to moths use cages with only male moths for treatments without oviposition (30 male S. exigua moths or 16 male M. sexta moths).

    Exposure of N. attenuata to S. exigua oviposition
    1. Whole N. attenuata plants are exposed in the flight cages to oviposition by the mated S. exigua moths (Figure 2B-C). Plants are placed individually in the cage by briefly opening it while taking care that moths do not escape.
      Note: S. exigua moths do not lay eggs on defined leaves of N. attenuata, however the moth oviposition behaviour depends on the plant species. Oviposition by S. exigua on defined leaf positions is possible, for example, on Solanum dulcamara plants of which selected leaves are exposed through slots at the cage sides as described below (see “Exposure of N. attenuata to M. sexta oviposition”).
    2. Overnight females lay eggs on the plants, but also on the pots and at the cage (Figure 2D-E). At the next morning, plants can be removed again individually by briefly opening the cage while taking care that the moths do not escape.
      Note: To place plants into the flight cages and remove them, a light should be placed behind the cage to attract the moths to the back side of the cage. As the moths may also remain on the plants and pots, at first the plant should be examined for moths, which are returned into the cage.
    3. All leaves are examined for egg clutches and single eggs (Figure 2F). Egg-laden leaves are labelled with hanging labels or a thread. The number of egg clutches, eggs and the leaf position where they are should be documented to first ensure that all eggs are removed before larval hatching and second to be able to explore the effect of these parameters on the larval performance and plant defence expression [see Bandoly and Steppuhn (2015)].
    4. All non-plant surfaces, i.e. pots and soil, need to be carefully inspected for eggs which are removed with a slightly moistened brush and may be collected in rearing boxes to retrieve larvae e.g., for rearing purposes.
    5. The time point of hatching can be estimated by regularly examining the eggs for darkening because the head capsule of the larvae starts to melanise shortly before they hatch (Figure 2G). Larvae will hatch after 2-4 d depending on the abiotic conditions in the greenhouse [most importantly temperature, e.g., it takes about 4 days at 25/15 °C (day/night)].
    6. Shortly before the larvae hatch, remove the eggs gently with a moistened brush to enable a standardised onset and extent of larval feeding as well as to ensure the treatment of oviposited plants without larval feeding. Again the eggs can be collected for further use e.g., for the larval feeding treatments.
      Note: Be careful not to damage the plant surface! If the egg attachment is very strong, try to loosen the eggs by applying water and try again after 1-2 min.


      Figure 2. Setup to expose Nicotiana attenuata plants to Spodoptera exigua oviposition. A. Four to five weeks old N. attenuata rosette stage plants. B. Mating S. exigua moths. C. Flight cage with plants and mated moths for oviposition. D. Female moth after oviposition on a leaf. E. Egg clutches on a plant pot. F. Different sizes and numbers of egg clutches on N. attenuata leaves. G. S. exigua eggs turning dark shortly before hatching. Photographs: Michele Bandoly

    Exposure of N. attenuata to M. sexta oviposition
    Note: As the allocation of defensive compounds is known to depend on leaf ontogeny (van Dam et al., 2001), it is best to standardise the leaf position that is exposed to oviposition, which is possible when the flight cage is prepared as such to bear slots at the sides of a cage in approximately the height that the designated leaf is situated on the plant in its pot (Figure 3A). The edges of the slots should be fixed with a seam so that the netting of the cage will not fray. The slots can be closed with hair clips.
    1. Determine the leaf positions in relation to the sink-source transition leaf (assimilate-import equals assimilate export), which can be estimated according to morphological characteristics as it unifies the characteristics of sink leaves, i.e. a light-green colouration and high trichome density, and that of source leaves, i.e. a dark-green colouration and low trichome density, to about equal extent (Figure 3B).
    2. Expose the second youngest source leaf of N. attenuata plants (see Figure 3B leaf +2) to M. sexta oviposition by inserting this leaf through slots in the cage that are located around the flight cage (Figure 3A, C).
    3. To avoid unnatural high egg loads, the oviposition cages have to be observed from dusk onwards, which is when these nocturnal moths become active. Though M. sexta moths lay single or two eggs on a plant under natural conditions, leaf material provided to moths in captivity usually receive overloads of eggs.
      Note: The light sources of the greenhouse should be kept off and to observe moth behaviour use a headlamp.
    4. As soon as an oviposition event occurred (Figure 3D), the respective leaf as well as that of the corresponding control plant are removed from the cages.
    5. Shortly before hatching, which can be estimated from the egg colour turning from light-green to white (Figure 3E), remove the eggs gently with a featherweight forceps. The natural egg incubation time of M. sexta is 4-5 d.
      Note: Be careful not to damage the plant surface! If the egg attachment is very strong, try to loosen the eggs by applying water and try again after 1-2 min.


      Figure 3. Setup to expose Nicotiana attenuata plants to Manduca sexta oviposition. A. Flight cage with mated M. sexta moths for oviposition on a defined leaf position of N. attenuata rosette stage plants. B. Positions and characteristics of sink and source leaves of N. attenuata in relation to the sink-source transition leaf. C. The +2 leaf (white arrow) is inserted through a slot at the side of the cage. D. Female M. sexta moths approaching the leaf exposed into the cage through a slot (white arrow) and during oviposition (black arrow) on a leaf exposed through a slot into the oviposition cage. E. The initially green M. sexta eggs turn white shortly before the larvae start hatching. Photographs: Michele Bandoly

  2. Larval performance (mortality, mass and developmental time) and feeding damage
    1. Select a defined leaf position (see Figure 3B) for larval application: either the second youngest source leaf (+2 leaf), a leaf position that is systemic to the leaf oviposited by S. exigua or M. sexta, or the local formerly egg-laden leaf if oviposition occurred on standardised leaves as described for M. sexta.
      Note: Due to the plant development during the egg incubation time the leaf positions in relation to the sink-source transition leaf have changed since the day of oviposition. The formerly egg-laden leaf corresponds now to leaf +3 or +4.
    2. Applicate similar numbers of neonate larvae to the selected leaf position of oviposited N. attenuata plants and the corresponding leaf position of control plants.
      1. As generalist S. exigua larvae feed gregariously and exhibit a high mortality on N. attenuata, apply 15-30 freshly hatched S. exigua larvae (dependent on availability) using a moistened brush (Figure 4A).
      2. The tobacco specialist M. sexta is feeding solitary or in low number on N. attenuata plants, so that not more than 3 larvae should be applied by grabbing the larvae with a featherweight forceps at their abdominal horn (Figure 4B).


        Figure 4. Application of Spodoptera exigua or Manduca sexta neonates. A. S. exigua larvae are applied using a moist brush. B. M. sexta larvae are applied with a featherweight forceps. C, D. Larvae are confined on the selected leaf using vented clip cages. Photographs: Michele Bandoly

    3. The larvae are confined on the selected leaf position using vented clip cages (see Figure 4C-D).
    4. Once per day carefully open the clip cages and examine the leaf as well as the cages for larvae. Count dead and alive larvae to determine larval mortality (in case of S. exigua a folding magnifier may be required to determine whether they are alive).
    5. To compare developmental times of larvae on oviposited and egg-free control plants also record their developmental stage when examining the larval mortality. In addition to the larval instar, you can also record three sub-instars of each instar to increase resolution (see also Figure 5 and 6A):
      1. Sub-instar A: Larvae shortly after moulting are characterised by a large head capsule in relation to the body.
      2. Sub-instar B: Larvae intensively feeding are characterised by a larger body volume in relation to their head capsule.
      3. Sub-instar C: Larvae preparing to moult are characterised by a tiny head capsule in relation to the body and are often lighter as they stop feeding.
    6. Larval weight can be measured every 2-3 d (in case of S. exigua, larvae can be first weighted after 3-4 d with a high precision balance of an accuracy of at least 0.1 mg). Again gently transfer small larvae with a moist brush.


      Figure 5. Spodoptera exigua larval instars and sub-instars. Freshly moulted (A), larvae in feeding stage (B) and larvae stopped feeding and prepare for moulting (C) are determined from different sizes of the dark brown head capsule in relation to the body diameter. Photographs: Stefanie Luka; Scale bar, 2 mm

    7. To warrant sufficient amounts of leaf material for the larvae to feed, the larvae are moved to the next younger leaf positions if required.
      Note: When larvae change instars take into account that larvae feed twice as much than previously. A transfer about every second day can be sufficient during the first week but thereafter larvae have to be transferred daily in longer lasting experiments.
    8. To assess feeding damage caused by the larvae photograph the leaves (without cutting them off) on a white board that is attached to a support stand with clamps that can be adjusted to the required height. The white board should have black reference areas on the four corners. Determine the fed leaf areas from the photographs in a graphic program [e.g., GNU Image Manipulation Program (GIMP) or Photoshop CS5 (Adobe Systems Incorporated, USA)] in relation to the reference areas.
    9.  To assess the antimicrobial activity in the haemolymph of M. sexta larvae as an immune parameter, larvae that were feeding for 6 days on oviposited or control plants are briefly chilled on ice and dabbed with a tissue soaked in 70% ethanol. Then the abdominal horn is cut off with sterilised scissors. The haemolymph is collected with a 10 µl-pipette while leaking out of the wound (Figure 6B). Immediately flash-freeze the haemolymph samples in 0.2 ml tubes in liquid nitrogen and analyse the antimicrobial activity with either a radial diffusion assay or micro-gel well diffusion assay or a microtiter broth dilution method as described in Du Toit and Rautenbach (2000).


      Figure 6. Developmental stages and haemolymph collection of Manduca sexta larvae. A. M. sexta larval instars and sub-instars. (A: Freshly moulted; B: Larvae in feeding stage; C: Larvae stopped feeding and prepare for moulting). B. Collection of haemolymph of young third instar M. sexta larvae from the horn that is cut off using a scissors. Photographs: Anke Steppuhn

  3. Standardised damage and harvest of leaf tissue for analyses of plant defence traits
    Note: Feeding-induced defensive compounds of N. attenuata such as caffeoylputrescine and protease inhibitor activity correlate positively with inflicted feeding damage (Bandoly et al., 2015). The feeding damage of S. exigua larvae on previously oviposited plants is lower than on control plants because of an increased larval mortality and a decreased larval growth. Thus, to compare induction levels of these compounds in egg-free and oviposited N. attenuata plants, plants of both treatment groups should be damaged to a comparable extent by either adjusting larval number and size or by mimicked larval herbivory.
    1. Standardisation of feeding damage by S. exigua larvae on oviposited and control N. attenuata plants
      1. Besides the experimental plants, an additional set oviposited and control plants hosting S. exigua larvae of the same egg batches as used for the experimental plants are used as supplier plants of larvae during the experiment.
      2. At least once per day experimental plants with and without prior oviposition are evaluated for the number and size of larvae. Differences between oviposited and control plants are adjusted by replacing dead or non-vital larvae with larvae of a corresponding size from the respective supplier plants.
      3. Ensure that the adjustment indeed resulted in equally damaged plants by determining the feeding damage from photographs as described above.
    2. Equal damage by mimicked herbivory (repeated wounding and application of larval oral secretions, as previously described e.g., in Halitschke et al., 2001).
      1. Oral secretions of third instar S. exigua larvae reared on N. attenuata leaf material are collected with a teflon tube connected to a 4 ml glass vial through the rubber septum in the lid (Figure 7A). The vial is kept on ice and connected through another tube in the lid with a vacuum pump. Provoke regurgitation by the larvae by gently nudging the larvae with the collection tube at the ventral side and collect the oral secretions using a vacuum slightly below atmospheric pressure (Figure 7B). The liquid fraction of the oral secretions after centrifugation in 1.5 ml tubes (at 4 °C) is diluted 1:1 with water for usage. (The oral secretions can be stored for a few days at -20 °C.) Keep oral secretions on ice during usage.
      2. Inflict a line of puncture wounds to each side of the mid-vein on a selected leaf position (e.g., +2 leaf; see above) of oviposited and egg-free control plants using a pattern wheel (Figure 7C) and immediately add 10 µl along the rows of puncture wounds using a pipette (Figure 7D). Repeat this treatment twice at the same leaves in intervals of 3 h.
      3. Repeat step C2b with the two next younger source leaves of oviposited and egg-free control plants on the next two days.


      Figure 7. Collection of oral secretions from Spodoptera exigua larvae to mimic the larval herbivory by mechanical wounding and application of oral secretions. Oral secretions from larvae are drawn into a glass vial connected to a vacuum pump (A) through a collection tube (B). C. Puncture wounds inflicted with a pattern wheel. D. Application of oral secretions of S. exigua to the wounds. Photographs: Anke Steppuhn (A, B) and Michele Bandoly (C, D)

    3. Harvest of leaf tissue
      1. To analyse plant defence compounds harvest the leaves exposed to herbivory or corresponding leaves of control plants (treatment II, IV in Box 1) with a scalpel and immediately flash-freeze the samples in 1.5 ml or 2 ml tubes (use a dissecting forceps to get the leaf material into the tube) in liquid nitrogen. Dependent on the analysed parameter, samples are taken within the first hours or days after herbivory treatment started. To assess the induction kinetics the same leaves may be repeatedly samples by harvesting parts of the leaves from the tip to the base as described [see Bandoly et al. (2015)].
      2. Analyse N. attenuata defence related compounds and regulators.
        1. Alkaloids and phenolics by high performance liquid chromatography (HPLC) according to Keinänen et al. (2001) 2-6 d after onset of herbivory.
        2. Protease inhibitor activity by a radial diffusion assay or a photometric assay in microwell plates as for example described in van Dam et al. (2001) and in Bandoly et al. (2015) 2-6 d after onset of herbivory.
        3. Phytohormones by liquid chromatography coupled to mass spectrometry (LC-MS) as for example described in Gaquerel et al. (2012) within the first hours after onset of herbivory.
        4. Defence gene expression by real-time qPCR as for example described in Bandoly et al. (2015) at a time point the respective gene is induced.

Data analysis

Example data for the effects of oviposition on the larvae, their feeding damage and the induction of plant defence parameters in the study systems described here as well as a statistical analyses of these data are presented in Bandoly et al. (2015) and Bandoly et al. (2016).

Acknowledgments

We would like to thank Roland Grichnik for aiding in the development of the bioassays with M. sexta described here. We also acknowledge the authors of previous studies that had established for example how to standardise leaf stages and analyse defence metabolites of N. attenuata of which this protocol could only cite examples. We are grateful for the financial support by the German Research Foundation (DFG; project B2 within the Collaborative Research Centre 973) and the German Federal Environmental Foundation (DBU), which supported Michele Bandoly with a stipend.

References

  1. Bandoly, M. and Steppuhn, A. (2016). A push-button: Spodoptera exigua oviposition on Nicotiana attenuata dose-independently primes the feeding-induced plant defense. Plant Signal Behav 11(1): e1114198.
  2. Bandoly, M., Grichnik, R., Hilker, M. and Steppuhn, A. (2016). Priming of anti-herbivore defence in Nicotiana attenuata by insect oviposition: herbivore-specific effects. Plant Cell Environ 39(4): 848-859.
  3. Bandoly, M., Hilker, M. and Steppuhn, A. (2015). Oviposition by Spodoptera exigua on Nicotiana attenuata primes induced plant defence against larval herbivory. Plant J 83(4): 661-672.
  4. du Toit, E. A. and Rautenbach, M. (2000). A sensitive standardised micro-gel well diffusion assay for the determination of antimicrobial activity. J Microbiol Methods 42(2): 159-165.
  5. Gaquerel, E., Steppuhn, A. and Baldwin, I. T. (2012). Nicotiana attenuata alpha-DIOXYGENASE1 through its production of 2-hydroxylinolenic acid is required for intact plant defense expression against attack from Manduca sexta larvae. New Phytol 196(2): 574-585.
  6. Halitschke, R., Schittko, U., Pohnert, G., Boland, W. and Baldwin, I.T. (2001). Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. III. Fatty acid-amino acid conjugates in herbivore oral secretions are necessary and sufficient for herbivore-specific plant responses. Plant Physiol 125: 711-717.
  7. Hilker, M., Schwachtje, J., Baier, M., Balazadeh, S., Baurle, I., Geiselhardt, S., Hincha, D. K., Kunze, R., Mueller-Roeber, B., Rillig, M. C., Rolff, J., Romeis, T., Schmulling, T., Steppuhn, A., van Dongen, J., Whitcomb, S. J., Wurst, S., Zuther, E. and Kopka, J. (2015). Priming and memory of stress responses in organisms lacking a nervous system. Biol Rev Camb Philos Soc.
  8. Keinanen, M., Oldham, N. J. and Baldwin, I. T. (2001). Rapid HPLC screening of jasmonate-induced increases in tobacco alkaloids, phenolics, and diterpene glycosides in Nicotiana attenuata. J Agric Food Chem 49(8): 3553-3558.
  9. Krügel, T., Lim, M., Gase, K., Halitschke, R. and Baldwin, I.T. (2002). Agrobacterium-mediated transformation of Nicotiana attenuata, a model ecological expression system. Chemoecology 12:177-183.
  10. Schoonhoven, L. M., van Loon, J. A. and Dicke, M. (2005). Insect-plant biology second edition. Oxford University Press. 421 pages (ISBN: 9780198525950)
  11. van Dam, N. M., Horn, M., Mares, M. and Baldwin, I. T. (2001). Ontogeny constrains systemic protease inhibitor response in Nicotiana attenuata. J Chem Ecol 27(3): 547-568.

简介

植物对具有不同防御反应的食草有反应(Schoonhoven等人,2005)。许多食草昆虫在它们的幼虫开始喂食之前将它们的卵沉积在它们的寄主植物上。因此,植物可以使用昆虫卵作为信号来增加它们对食草动物的抗性。在这里,我们报告实验程序,以探究是否和如何昆虫产卵影响植物抵抗喂养幼虫。所描述的方法揭示了先前暴露于鳞翅目蛾产卵的烟草(Nicotiana attenuata)( N。attenuata )植物通过万古霉属(Spodoptera exigua) ( S.exigua )和专家 ( sexta )幼虫与防御反应的诱导增加,或幼虫的免疫状态(Bandoly等人,2015; Bandoly等人,2016)。因此,昆虫产卵可以引发进食诱导的植物防御(引发:根据先前刺激的经验增强的植物对胁迫的反应; Hilker等人,2015)。使用用于昆虫产卵和幼虫食草的标准化程序的全因子实验允许破译植物暴露于昆虫卵对幼虫性能,进食损伤和免疫状态的影响以及区分卵诱导的植物对卵发生反应的响应到幼虫喂养。

材料和试剂

  1. 反应管,0.2ml(例如,Carl Roth GmbH + Co.KG,目录号:H560.1)
  2. 反应管,1.5ml或2ml(例如Carl Roth GmbH + Co.KG,目录号:CK06.1)
  3. Nicotiana attenuata Torr。 例如。 沃森(茄科),4-5周龄(玫瑰花阶段)
  4. Spodoptera exigua Hübner(夜蛾科,鳞翅目)或 Manduca sexta Linnaeus(Sphingidae,Lepidoptera)
  5. 液氮
    注意:所使用的所有材料和设备都是一般的实验室设备或家用器皿,我们只提到我们使用的示例产品。

设备

  1. 一般设备
    1. 软漆刷[例如,Rotmarder 122A,尺寸2(Habico,目录号:50122A10)]
    2. 羽毛钳(例如,Carl Roth GmbH + Co.KG,目录号:AN00.1)
    3. 收纳箱( 例如,14 x 21 x 5厘米),用纱布(尼龙网0.12毫米宽)盖子
    4. 精确平衡(例如,Sartorius AG,型号:Sartorius MC210S)

  2. 植物上的植物
    1. 飞行笼[例如,,kweekkooi,60 x 60 x 90厘米(Vermandel,目录 编号:80.304)或屈肌,42×42×76cm(Rolf C.Hagen Inc.,Exo Terra,型号:PT2552)]
      注意:飞行笼最终有插槽 限定的叶片可以通过其露出的保持架侧部 用小爪发夹闭合。
    2. 悬挂标签[例如,Hängeetiketten,HERMA FACHSHOP,型号:6901]]或线程
    3. 前灯(例如,Petzl,型号:PIXA ® 3)

  3. 幼虫性能,进食损伤和血淋巴取样
    1. 通风的夹子笼[例如,由两个敷料杯(,例如,Ø7.03,2.3 cm(KIV-KREIS,目录号:770405509))   边缘,一个杯子的底部由纱布(尼龙网0.12mm 宽度),并且笼子可以用小爪发夹闭合]
    2. 折叠放大镜[例如,TRIPLET 20x,21 mm(Light In The Box Ltd.,目录号:01239576)]
    3. 实验室支架与夹具
    4. 带参考区域的白板(例如,黑色方块为1 x 1厘米的白纸叠层)
    5. 使用EFS 18-25mm微距镜头(Canon Inc.,型号:Canon EF-S 18-55mm f/3.5)的相机[例如,Canon EOS 1200D(Canon Inc.,型号:Canon EOS 1200D) -5.6 IS II)]
  4. 标准化的损伤和叶组织的收获,用于植物防御性状的分析
    1. 真空泵(例如,,Vacuubrand,型号:PC 510)
    2. 聚四氟乙烯管[例如外径1.6mm(VWR International,目录号:BOHLS1810-01)]
    3. 具有橡胶隔膜盖的玻璃小瓶[例如,4ml HPLC小瓶(Techlab,目录号:FLK 101.243和FLK 101.972)]
    4. 跟踪/图案轮(例如,Nähmit,目录号:070210)
    5. 10μl移液器
    6. 手术刀(例如,Carl Roth GmbH + Co.KG,目录号:X004.1)
    7. 解剖钳(例如,Carl Roth GmbH + Co.KG,目录号:2690.1)

程序

  1. 植物上的植物
    1. 排序。 exigua 或 M。 sexta 蛹的性别。 图1描述了 男性和女性鳞翅目之间的歧视特征 蛹。
    2. 让蛹在用于的飞行笼中关闭 产卵。 在 S的情况下。 exigua 将15个女性和15个雄性蛹放置在a   飞行笼。 在 M的情况下。 sexta 将8个女性和8个男性蛹放在a 飞行笼。 每天检查笼子两次(在早晨和在 晚上)为eclosed飞蛾。 以确保飞蛾有时间交配 并已开始下鸡蛋使用笼子2 d后eclosion 在 S的情况下产卵。 exigua 和之后3天。sexta 。


      图1.雄性(A)和雌性(B)草地贪夜蛾蛹和雄性(C)和雌性(D)玉米蚜虫蛹的腹部。/strong>箭头表示腹侧的生殖器结构 不同性别之间的形态和他们所在的部分。 照片:Stefanie Luka(A,B),Anke Steppuhn(C,D)。比例尺,2 mm(A,B),5mm(C,D)

    3. 均衡的微小差别 处理组之间的植物个体发育,4-5周龄的烟草 (如在Krügel等人(2002)中所述生长的;图2A]大小(即莲座直径)和伸长状态 (茎发育程度)并且将相等的植物分配给所有处理 的每个生物复制。对于全因子实验设计 能够评价产卵对植物防御的影响 需要参数四个治疗组(框1)
      Box1。完全因子启动实验所需的处理
      I.天然卵孵化时间后的产卵和幼虫喂食
      II。产卵不受幼虫喂食
      III。未经产卵的幼虫喂养
      IV。控制没有产卵和幼虫进食的植物

    4. 控制实验过程和其他刺激 与暴露于蛾相关联使用只有雄蛾的笼子 处理而不产卵(30只雄性。ua或16只雄性M)。 sexta飞蛾)。

    曝光 N。 attenuata 到 S。 exigua oviposition
    1. 全部。 衰减植物在飞行笼中暴露由交配的S产卵。 exigua 蛾(图2B-C)。植物是 通过短暂打开它,同时小心地单独放置在笼子里 那飞蛾不逃脱。
      注意:S. exigua蛾不产卵 定义的叶片,但是蛾的产卵行为 取决于植物物种。 S. exigua在定义叶上的转位 位置是可能的,例如,在Solanum dulcamara植物上  所选的叶通过在笼侧的槽暴露 (参见"Exposure ofN.λfplatato M. sexta 产卵")。
    2. 过夜女性在植物上产卵,但也  在罐和笼上(图2D-E)。第二天早上,植物 可以通过短暂打开笼子再次单独移除 注意飞蛾不逃脱。
      注意:放置植物 进入飞行笼,并删除它们,一个光应放在后面 该笼子将该蛾子吸引到笼子的背面。作为蛾也可能留在植物和花盆上,首先植物应该 检查飞蛾,将其返回笼中。
    3. 所有叶子 检查鸡蛋离合器和单个鸡蛋(图2F)。 蛋载 叶子用吊标记或螺纹标记。 鸡蛋的数量 离合器,鸡蛋和叶的位置,应该记录在案   首先确保在幼虫孵化之前除去所有的卵 二是能够探索这些参数的效果 幼虫表现和植物防御表达[参见Bandoly和 Steppuhn(2015)]。
    4. 所有非植物表面,即盆和土壤, 需要仔细检查与a一起去除的鸡蛋 轻微润湿的刷子,并可以在饲养箱中收集 检索幼虫例如,用于饲养目的
    5. 时间点 孵化可以通过定期检查鸡蛋变暗来估计 因为幼虫的头囊在不久之前开始变黑  它们孵化(图2G)。幼虫会在2-4天后孵化,取决于 温室中的非生物条件[最重要的是温度,例如,在25/15℃(白天/夜晚)需要约4天]。
    6. 不久之前 幼虫孵化,用湿润的刷子轻轻地去除鸡蛋 使幼虫进食的标准化起始和程度以及 确保处理产卵的植物没有幼虫进食。再次 可以收集蛋以供进一步使用,例如,用于幼虫进食 治疗 注意:小心不要损坏植物表面!如果  鸡蛋附着很强,尝试通过应用水来松开鸡蛋  并在1-2分钟后重试。


      图 。衰减阶段植物。 B.配合。 exigua 蛾。 C.具有植物和配合的飞蛾的飞行笼。 D.在叶片上产卵后的雌蛾。鸡蛋在植物罐离开。 F.不同尺寸和数量的鸡蛋离合器在N上。衰减叶。 G. S。 exigua 鸡蛋在孵化前不久变暗。照片:Michele Bandoly

    曝光 N。 attenuata 到 M。 sexta 产卵
    注意:由于已知防御性化合物的分配取决于叶子个体发育(van Dam et al。,2001),因此最好将暴露于产卵的叶片位置标准化,这是在飞行笼制备成在笼子的侧面在大约指定叶片位于其盆中的植物(图3A)的高度处具有槽。槽的边缘应用接缝固定,以便保持架的网不会磨损。插槽可以用发夹关闭。
    1. 确定相对于汇源的叶位置 过渡叶(同化进口等于同化出口),可以 根据其统一的形态特征进行估计 水槽叶的特征,即浅绿色的颜色高毛状体密度和源叶的密度,即暗绿色 着色和低毛发密度,达到大致相等的程度(图3B)
    2. 暴露 N的第二个最新的源叶。 (参见 图3B叶+2)到M。 sexta 产卵 穿过位于飞行笼周围的笼中的狭槽 (图3A,C)。
    3. 为了避免不自然的高蛋负荷, 产卵笼必须从黄昏向前,这是什么时候观察 这些夜间飞蛾变得活跃。 虽然 M。 sexta 蛾类单一或   两个鸡蛋在自然条件下的植物上,叶材料提供给   蛾的蛾通常会收到蛋的过多 注意:温室的光源应该保持关闭,并观察蛾的行为使用头灯。
    4. 一旦产卵事件发生(图3D),相应的叶以及相应对照植物的叶 从笼子。
    5. 孵化前不久,可以估计 从鸡蛋颜色从浅绿色转变为白色(图3E), 用轻量级镊子轻轻取出鸡蛋。天然鸡蛋 孵育时间为M. sexta 是4-5天。
      注意:小心不要损坏  植物表面!如果鸡蛋附着很强,尝试松开 使用水,并在1-2分钟后再试一次。


      图3.将 Nicotiana attenuata 植物暴露于 产卵的设置 sexta 蛾  N的定义的叶位置。衰减阶段植物。乙。 N的源和叶的位置和特征。衰减相对于信宿源过渡叶的衰减。 C. +2叶(白色 箭头)插入通过在笼的侧面的槽。 D. Female M. sexta飞蛾接近通过槽暴露于笼中的叶 (白色箭头)和在产卵期间(黑色箭头)暴露的叶通过一个槽进入产卵笼。 E.最初的绿色M。 sexta 鸡蛋在幼虫开始孵化前不久就会变白。 照片:Michele Bandoly

  2. 幼虫的性能(死亡率,质量和发育时间)和进食损伤
    1. 为幼虫选择一个定义的叶位置(见图3B) 应用:第二最新源叶(+2叶),叶 位置,其对于由S产生的叶是全身的。 exigua 或 M。 sexta ,或者如果产卵,则为当地以前的含蛋黄叶 如对于M所描述的标准化叶。 sexta 。
      注意:由于 植物发育期间鸡蛋孵化时叶的位置 与源 - 源过渡叶的关系自从一天以来已经改变的产卵。 以前的含蛋叶现在对应叶+3 或+4。
    2. 应用相似数量的新生幼虫 选择的产卵的叶位置。 衰减植物和 对照植物的相应叶位置。
      1. 作为通才。 exigua 幼虫饲料,并且在N上表现出高的死亡率。 attenuata ,应用15-30新孵出的 S。 exigua 幼虫 可用性)使用润湿刷(图4A)
      2. 烟草 专家。 sexta 衰减植物,使得通过抓取不应超过3只幼虫 幼虫在他们的腹角有羽毛镊子(图 4B)。


        图4. Spodoptera exigua 或 Manduca的应用   sexta 新生儿。 A. exigua 幼虫。 B. M。 sexta 幼虫应用羽毛钳。 C.,D.Larvae 使用通气夹笼限制在选定的叶。 照片: Michele Bandoly

    3. 使用通气的夹笼将幼虫限制在选定的叶位置(参见图4C-D)。
    4. 每天一次小心地打开夹笼,检查叶 以及作为幼虫的笼子。 计数死亡和活着的幼虫来确定 幼虫死亡率(在放线虫的情况下可以是) 需要确定他们是否活着)。
    5. 比较 发育时期的幼虫对产卵和无卵的对照植物 也记录了它们在检查幼虫时的发育阶段 死亡。 除了幼虫的时代,你还可以记录三个 每个阶段的子阶段以增加分辨率(也参见图5和图5)   6A):
      1. 子阶段A:蜕皮后不久的幼虫的特征是相对于身体有大的头囊。
      2. 子龄B:幼虫集中喂养的特征在于相对于它们的头囊具有更大的体积。
      3. 子龄C:幼虫准备换羽的特点是一个小小的 头囊相对于身体,并且通常在它们停止时更轻 喂养。
    6. 幼虫重量可以每2-3天测量一次(在情况下)。   exigua ,幼虫可以首先在3-4天后以高精度进行加权 平衡精度至少为0.1mg)。 再轻轻地转移小 幼虫用湿毛刷。


      图5. 草地夜蛾幼虫龄期和亚龄。新鲜蜕皮(A),幼虫进食 阶段(B)和幼虫停止喂养并准备脱壳(C) 由不同尺寸的深棕色头囊决定 与体直径的关系。 照片:Stefanie Luka; 比例尺,2 mm

    7. 保证足够数量的叶材料 幼虫进食,幼虫移动到下一个更年轻的叶位置如果需要。
      注意:当幼虫变化时考虑 幼虫的饲料是以前的两倍。关于每一个的转移 第二天可以在第一周足够,但后来幼虫  必须每天转入更持久的实验。
    8. 至 评估由幼虫照片所造成的进食损伤叶片 (不切断它们)在附接到a的白板上 支撑架,带有可以调节到所需高度的夹具。 白板应在四个角上具有黑色参考区域。 在图形程序中从照片中确定进料的叶面积 [例如,GNU图像处理程序(GIMP)或Photoshop CS5(Adobe Systems Incorporated,USA)]相对于参考区域
    9.  以评估M的血淋巴中的抗微生物活性。 sexta幼虫作为免疫参数,饲养6天的幼虫 产卵或对照植物在冰上短暂冷却并用a组织浸泡在70%乙醇中。然后切除腹角 消毒剪刀。用10μl移液管收集血淋巴 同时泄漏出伤口(图6B)。立即闪烁冻结  血淋巴样品在0.2ml管中的液氮中并分析 抗微生物活性与径向扩散测定或微凝胶  孔扩散测定法或如所述的微量滴定肉汤稀释法  in Du Toit and Rautenbach(2000)。


      图6. Developmental  阶段和血淋巴收集 。 sexta 幼虫龄期和亚龄。 (A:新鲜蜕皮; B:幼虫 喂养阶段; C:幼虫停止喂养并准备蜕皮)。乙。 收集年轻三龄的血淋巴。 sexta 幼虫 使用剪刀切掉的角。照片:Anke Steppuhn

  3. 标准化的损伤和叶组织的收获,用于植物防御性状的分析 注意:进食诱导的淡褐链球菌的防御化合物如咖啡酰腐胺和蛋白酶抑制剂活性与受到的进食损伤正相关(Bandoly等人,2015)。 S. exigua幼虫对先前产卵的植物的摄食损伤比对照植物低,因为幼虫死亡率增加和幼虫生长减少。因此,为了比较这些化合物在无蛋和成熟的淡褐叶枯萎病植物中的诱导水平,两个处理组的植物应通过调整幼虫数量和大小或通过模拟幼虫食草来损害相当程度。
    1. 进食损伤的标准化。 exigua 幼虫对照和对照。衰减植物
      1. 除了实验植物,另外一组oviposited和 控制植物托管。 exigua 幼虫 为实验植物用作幼虫的供应植物  本实验。
      2. 每天至少一次实验植物 并且没有先前产卵的数量和大小 幼虫。调节产卵和对照植物之间的差异 通过用相应的幼虫代替死亡或非重要的幼虫尺寸从相应的供应商工厂。
      3. 确保 调整确实导致了同样受损的植物 如上所述从照片馈送损伤。
    2. 平等伤害   通过模仿草食(反复伤口和应用幼虫口服   分泌物,如先前在例如Halitschke等人中描述的,,2001)。
      1. 第三龄的口腔分泌物。 exigua 幼虫。 用连接到4的特氟龙管收集衰减的叶材料   ml玻璃小瓶通过盖子中的橡胶隔膜(图7A)。 的 小瓶保持在冰上并通过盖中的另一个管与a连接真空泵。通过轻轻地推动幼虫诱导反流 幼虫与收集管在腹侧并收集口服  分泌物使用的真空度略低于大气压(图 7B)。离心后的口腔分泌物的液体部分 将1.5ml管(在4℃下)用水1:1稀释以备使用。 (口头 分泌物可以在-20℃下储存几天。)保持口腔分泌物  在使用期间的冰。
      2. 对每个人造成一条穿刺伤口线 在选择的叶位置(例如,,+ 2叶;参见图1)上的中间静脉侧 以上)的产卵和无蛋对照植物使用图案轮 (图7C),并立即沿着穿刺伤口的行添加10μl (图7D)。重复这个处理两次 叶间隔3 h。
      3. 重复步骤C2b与两个下一个年轻的源叶的无卵和无卵的对照植物在接下来的两天。


      图7.从灰翅夜蛾(Spodoptera exigua)幼虫中收集口服分泌物 通过机械创伤和应用来模仿幼虫食草 口腔分泌物。幼虫的口服分泌物被吸入玻璃小瓶中   通过收集管(B)连接到真空泵(A)。 C。 穿刺伤口造成图案轮。 D.口服的应用 分泌物。 exigua 到伤口。 照片:Anke Steppuhn(A,B)   和Michele Bandoly(C,D)

    3. 叶组织收获
      1. 至 分析植物防御化合物收获暴露于食草的叶 或对照植物的相应叶(处理II,方框1中的IV)用解剖刀立即快速冷冻样品在1.5 ml或2 ml管(使用解剖镊子获得叶材料 管)在液氮中。 取决于分析的参数,样品 是在食草治疗后的最初几小时或几天内服用的 开始。 为了评估诱导动力学,可以使用相同的叶 通过从叶尖收集部分叶子重复样品   基础(参见Bandoly等人(2015)]。
      2. 分析 N。 衰减抗原相关化合物和调节剂。
        1. 碱性和酚类通过高效液相色谱 (HPLC)根据Keinänen等人(2001)2-6天后开始 草食
        2. 蛋白酶抑制剂活性通过径向扩散 测定或在微孔板中的光度测定描述在van Dam等人(2001)和Bandoly等人(2015)2-6d中。 在食草发病后。
        3. 液体的植物激素 色谱偶联至质谱(LC-MS) 在Gaquerel等人(2012)中描述的发作后的最初几小时内 的食草
        4. 防御基因表达的实时qPCR为 在Bandoly等人(2015)中描述的示例 诱导各基因。

数据分析

在此描述的研究系统中对产卵对幼虫的影响,它们的摄食损伤和植物防御参数的诱导的实例数据以及这些数据的统计分析在Bandoly等人的 (2015)和Bandoly 等人(2016)。

致谢

我们要感谢Roland Grichnik帮助用em开发生物测定。 sexta 。我们还承认以前的研究的作者已经建立了例如如何标准化叶阶段和分析N的防御代谢物。衰减,其中该协议只能举出例子。我们感谢德国研究基金会(DFG;合作研究中心973内的B2项目)和德国联邦环境基金会(DBU)的资金支持,该基金会支持Michele Bandoly的津贴。

参考文献

  1. Bandoly,M。和Steppuhn,A。(2016)。 按钮: Spodoptera exigua 在 Nicotiana attenuata上产卵剂量独立地补充饲养诱导的植物防御。植物信号行为 11(1):e1114198。
  2. Bandoly,M.,Grichnik,R.,Hilker,M.and Steppuhn,A.(2016)。 由昆虫产卵引起的抗食草动物防御: 通过草食动物特异性效应。 植物细胞环境39(4):848-859。
  3. Bandoly,M.,Hilker,M。和Steppuhn,A。(2015)。 由 Nicotiana attenuata 的 Spodoptera exigua 进行的转换原生动物诱导植物防御幼虫草食性。植物J 83(4):661-672。
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  5. Gaquerel,E.,Steppuhn,A.and Baldwin,I.T。(2012)。通过其生产2-羟基亚麻酸的 艾草提取物 alpha-DIOXYGENASE1是需要用于完整植物防御表达对来自 Manduca sexta 幼虫的攻击。<​​/a>新植物 196(2):574-585。
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引用:Bandoly, M. and Steppuhn, A. (2016). Bioassays to Investigate the Effects of Insect Oviposition on a Plant’s Resistance to Herbivores. Bio-protocol 6(11): e1823. DOI: 10.21769/BioProtoc.1823.
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