by Kelly F. Oten1, Allen C. Cohen2, John B. Strider1, and Fred P. Hain1

Abstract

This study uses scanning electron microscopy (SEM), a surface-imaging technique, to investigate feeding behaviors of the hemlock woolly adelgid (HWA), Adelges tsugae Annand. Understanding feeding behavior directly relates to the pest-host interactions which can elucidate resistance mechanisms in hemlocks. Salivary sheath material, a viscous product of the salivary glands that hardens upon extrusion, was imaged; insertion site is on the adaxial side of the needle, below the abscission layer and no major differences were noticed between the western population HWA and eastern population HWA. Imaging of stylets within the plant tissue was unsuccessful using SEM and further investigations will incorporate other techniques.

Authors: Kelly F. Oten1, Allen C. Cohen2, John B. Strider1, and Fred P. Hain1

  1. Department of Entomology, North Carolina State University, Raleigh, NC
  2. Insect Diet and Rearing Research, LLC, Raleigh, NC

Keywords

Adelges tsugae, feeding behavior, host resistance, scanning electron microscopy

Introduction

The hemlock woolly adelgid (HWA), Adelges tsugae Annand, has caused massive mortality of hemlocks in eastern North America over the last few decades. After becoming infested, trees can die in as little as four years (McClure 1987, 1991). However, not all trees succumb to the infestations. The seven hemlock species occurring in the native range of HWA exhibit varying levels of resistance (Table 1). This resistance is attributed to innate resistance, a complex of natural enemies, and the scattered distribution of hemlocks (McClure 1992, Montgomery and Lyon 1996). Until recently, literature has stated that eastern hemlock (Tsuga canadensis (L.) Carriére) and Carolina hemlock (T. caroliniana Engelm.) are the only hemlock species susceptible to HWA. However, following massive devastation of hemlock stands throughout the Appalachian Mountains, reports of individuals and stands surviving or avoiding infestations altogether are surfacing. There is discrepancy between environmental conditions and innate resistance being the cause of such cases. Investigations into mechanisms of resistance are prompted by two observations: resistance varies between the nine species of hemlock that occur worldwide, and resistance varies within eastern and Carolina hemlocks. Therefore, the previously accepted notion of comprehensive susceptibility of eastern and Carolina hemlock is becoming uncertain.

Host plant resistance is an important part of agricultural integrated pest management (IPM) and it has been long accepted that tree resistance should be incorporated into forest management as well (Stark 1965, Larsson 2002). A fundamental part in understanding resistance is a thorough knowledge of pest-host interactions, of which insect feeding behavior is a major component (Painter 1951, Davis 1985). Studying the feeding behavior of HWA is an essential stepping stone in reaching a resistant variety of hemlock.

Table 1. Resistance levels and native ranges of hemlocks.

Hemlock SpeciesRangeHWA Resistance
Eastern hemlock
Tsuga canadensis
Eastern North AmericaVaries
(Susceptible to moderately resistant)
Carolina hemlock
T. caroliniana
Southern AppalachiansVaries
(Susceptible to moderately resistant)
Chinese hemlock
T. chinensis
ChinaHighly resistant
Northern Japanese hemlock
T. diversifolia
Northern Japan
(high elevations)
Resistant
Western hemlock
T. heterophylla
Western North AmericaModerately resistant
Mountain hemlock
T. mertensiana
Western North AmericaModerately resistant
Southern Japanese hemlockSouthern Japan
(low elevations)
Moderately resistant
Himalayan hemlock
T. dumosa
Himalayan MountainsModerately resistant
Forrest’s hemlock
T. forrestii
ChinaModerately resistant

A large part of information regarding the feeding behavior of HWA is based on aphids as a model. Aphids and HWA have similar mouthpart form and function; four stylets (two maxillary and two mandibular) make up the stylet bundle. This stylet bundle forms two channels, the food and salivary canals, and is inserted into the plant to feed. Both insects produce watery saliva, extruded as an aid in feeding, and sheath saliva, which surrounds the stylet bundle (Miles 1999). However, unlike aphids, HWA is stationary and do not feed on phloem. Rather, HWA feeds on the ray parenchyma cells, starch-filled storage cells in the xylem (Young et al. 1995). It is unknown whether extra-oral digestive enzymes, such as amylase, are used to digest these starches prior to ingestion. The sheath material, which is extruded in a bead-like manner as the adelgid explores the host surface, likely plays multiple important roles: 1) to assist in the sucking mechanisms by closing off the ingestion mechanism, preventing leaks, 2) to provide a mechanical advantage in stylet bundle stabilization providing leverage-fulcrum points, 3) to allow easy stylet bundle reinsertion following each molt, 4) to provide better movement through or between cell walls, and 5) protect or insulate the insect’s mouthparts against plant defenses. The sheath material is very stable and remains on and in the plant when the insect is removed.

Analysis of stylet pathways may help elucidate mechanisms of resistance (Spiller et al. 1985). Relationships of stylet pathways were also shown to be useful in assessing relative susceptibility of host plants (Cohen et al. 1996, Cohen et al. 1998). Relative susceptibility of different cultivars of cotton was also demonstrated in whiteflies by studying patterns of stylet pathways (Chu et al. 1999). In chinch bugs feeding on resistant cultivars of St. Augustine grass, the sheath material was more abundant than in susceptible cultivars (Rangasamy et al. 2009). In another study, aphids took three times as long to locate internal feeding sites in resistant soybean cultivars (Crompton and Ode 2010). It appears the detection of internal feeding sites is more difficult in resistant plants for these hemipteran insects, evidence which can provide insights to HWA feeding studies. Our hypotheses are that the stylets of HWA will branch more within resistant hemlocks and physical and/or chemical differences in the host surface will contribute to HWA’s host selection.

Methods

Infested hemlock material was collected from Laurel Springs and Crossnore, NC. Branches were kept in buckets of water in an incubator kept at 16° C and 80% humidity. Small sections of infested branchlets were freeze-fractured, freeze-dried, and mounted for scanning electron microscopy (SEM) observation. SEM is a valuable tool capable of high resolution at high magnifications. It is helpful in observing external stylet habits; fracturing techniques were used in combination as an endeavor to reveal internal stylet movement.

Material from the western population of HWA was sent on ice via overnight shipping by Glenn Kohler (Washington State Department of Natural Resources). Infested branches were kept in quarantine at the Beneficial Insects Lab in Cary, NC. Infested material was fixed in 3% glutaraldehyde prior to removing from quarantined area. Material was post-fixed in 1% osmium tetroxide, dehydrated in a critical point dryer, and prepared for SEM observation.

Results and Discussion

Several interesting finds were noted at the high magnification SEM provided. Morphological differences between the crawler stage and the adult stage were observed (Fig. 1). Aside from being smaller (~0.5 mm), crawlers lacked wax pores, had prominent antennae, and the stylet bundle retracted. Conversely, adults were approximately 1 mm in size, showed prominent pores for wax extrusion, had reduced antennae, and stylets extended. Reduction in antennae is likely related to its transition from the host-seeking stage of the insect. Future work should be done to determine sensory function of the antennae, as they have distinctive morphological characters, such as a pit with finger-like projections and long setae (Fig. 2).

Insertion points of the stylet consistently occurred below the abscission layer of the needle on the adaxial side of the needle (Fig. 3). This is consistent with the work done by Young et al. (1995). It was also observed that sheath material was produced prior to the stylet bundle’s contact with the host surface. This external production allows us to determine the size and structure we expect to find within the plant material. A break in the sheath material reveals the internal stylets and the bumpy texture of the material is an indicative character (Fig. 4).

Figure 1

Figure 1

Figure 2

Figure 2

Figure 3

Figure 3

Figure 4

Figure 4

HWA from the western population were slightly larger and rounder than the eastern population, but there were no major morphological differences noticed. Insertion site between the two populations were alike. Further investigations will include observations of wood anatomy of western and mountain hemlocks.

Parenchyma cells were imaged, but sheath material within the plant tissue was never observed within them. Further investigations into host resistance and feeding behavior will include clearing of hemlock material and staining of sheath material followed by light and confocal microscopy, further SEM of plant material using cryo-SEM, and anatomical studies of the wood of different hemlock species. In addition to observing physical characteristics of different hemlock species, chemical components of the foliar wax will be studied.

Acknowledgements

This research was supported by the USDA Forest Service. We extend thanks to Valerie Knowlton and John MacKenzie from the Center for Electron Microscopy at North Carolina State University, to Rebecca Norris and Kathy Kidd from the Beneficial Insects Lab, to Glenn Kohler for providing multiple samples of western populations of HWA, and to Robert Jetton, JC Domec, Jerry Moody, John Frampton, Micah Gardner, and the staff at the Upper Mountain Research Station in Laurel Springs, NC.

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