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1
Naturwissenschaften (2008) 95: 433-441
Received: 26 October 2007 / Revised: 3 January 2008 / Accepted: 12 January 2008
The original publication is available at www.springerlink.com as Naturwissenschaften 2008 On Line First DOI
10.1007/s00114-008-0346-3
ORIGINAL PAPER: Author copy
Unique zinc mass in mandibles separates drywood termites from other
groups of termites
Bronwen W. Cribb,
*,1,2
Aaron Stewart,
2,3
Han Huang,
4
Rowan Truss,
4
Barry Noller,
3
Ronald Rasch
1
, Myron P. Zalucki
2
1
Center for Microscopy & Microanalysis, The University of Queensland, Brisbane, 4072, Australia.
2
School of Integrative Biology, The University of Queensland, Brisbane, 4072, Australia.
3
National Research Centre for Environmental Toxicology, 39 Kessels Road, Coopers Plains, Brisbane, 4108,
Australia.
4
School of Engineering, The University of Queensland, Brisbane, 4072, Australia.
*
To whom correspondence should be addressed
E-mail: b.cribb@uq.edu.au
Phone: +61 7 33657086
Fax: +61 7 33463993
Abstract Previously the presence of metals in arthropod mandibles has been linked with
harder cuticle and in termites a 20% increase in hardness has been found for mandibles
containing major quantities of zinc. The current study utilizes electron microscopy and
Energy Dispersive X-ray microanalysis to assess incidence and abundance of metals in all
extant subfamilies of the Isoptera. The basal clades contain no zinc and little to no
manganese in the cutting edge of the mandible cuticle, suggesting that these states are
ancestral for termites. However, experimentation with mandibles in vitro indicates the
presence of some elements of the cuticular biochemistry necessary to enable uptake of zinc.
The Termopsidae, Serritermitidae, Rhinotermitidae and Termitidae all contain minor
quantities of manganese, while trace to minor quantities of zinc occur in all except the
Serritermitidae. In contrast, all Kalotermitidae or drywood termites contain major levels of
zinc in the mandible edge. Diet and life type are explored as links to metal profiles across
the termites. The presence of harder mandibles in the drywood termites may be related to
lack of access to free water with which to moisten wood. Scratch tests were applied to a set
of mandibles. The coefficient of friction for Cryptotermes primus (Kalotermitidae)
mandibles, when compared with species from other subfamilies, indicates that zinc
containing mandibles are likely to be more scratch resistant.
Key words Insect, biomineralization, jaws, EDX, EDS
Introduction
A range of metals including zinc (Zn) and manganese (Mn) are found in insects and other
arthropods concentrated within the cuticle (or exoskeleton) of mandibles, mouth hooks,
claws and ovipositors (Hillerton and Vincent 1982; Hillerton et al. 1984; Quicke et al.
1998; Fontaine et al. 1991; Schofield 2001; Lichtenegger et al. 2003; Morgan et al. 2003;
Schofield 2005; Birkedal et al. 2006). A number of studies on insects establish links
2
between Zn enrichment and enhanced mechanical properties such as hardness, for example
in grasshoppers, Schistocerca gregaria and Locusta migratoria (Orthoptera) and the leaf-
cutter ant, Atta sexdens rubipilosa (Hymenoptera) (Edwards et al. 1993; Schofield et al.
2002; Hillerton et al. 1982). Zn in the jaws has also been linked with increased local
hardness (and stiffness) in the marine polychaete worm Nereis limbata (Lichtenegger et al.
2003). In termites the metals, Zn and Mn, occur only in the edge of the mandibles and not
below in the general cuticle of the mandible (Cribb et al. 2007). Recently it was found that
the mandible edges of termites which contain major quantities of Zn are 20% harder than
mandibles without this metal (Cribb et al. 2007). Interestingly, of the six species of termite
investigated for hardness in this study, the two species found to contain Zn were from the
same group, the Kalotermitidae or drywood termites. Beyond the six species analysed in
that study only four other species of termites have been investigated for metals in
mandibles and while some show minor quantities of Mn but no Zn (Fawke et al. 1997;
Yoshimura et al. 2002, 2005) the two others are drywood termite species and show Zn in
the mandible edge (Ohmura et al. 2007).
Since Zn enriched mandibles are substantially harder, their presence may relate to
capacity to inflict damage to wood and hence pest status. Presence may also relate to
specific life type characteristics. The question arises as to the distribution of Zn occurrence
within the termites. In the current study we survey all extant subfamilies in the Isoptera:
the Mastotermitidae, Hodotermitidae, Termopsidae, Serritermitidae, Rhinotermitidae, and
Termitidae. It should be noted that we have chosen to maintain the classification schema
used prior to Inward et al. (2007) due to the as yet unresolved debate initiated by Lo et al.
(2007).
Materials and Methods
Samples were assessed for elemental composition using a JEOL JSM 6460 LA low vacuum
analytical scanning electron microscope equipped with an integrated JEOL Hyper mini-
cup, 133eV resolution, ultra thin window (UTW), SiLi crystal, Energy Dispersive X-ray
Spectrometer (EDS) following Cribb et al. (2007). X-ray analysis was carried out in low
vacuum mode to obviate a metal coating. Standardless quantitation software comprised the
JEOL Analysis Station (V3.51). Acquisition conditions on the SEM were 20kV, 10 mm
working distance and 30 sec live time acquisition at approximately 10-15% dead time.
Because no suitable standard is available to match the material being assessed, standardless
quantitation was used with a phi-rho-z correction and elemental mass reported qualitatively
as trace (<0.5 mass % dry weight), minor (a few percent) or major (>5 - ≥20 % mass
weight) (following Newbury 1991).
Samples from all extant subfamilies of the Isoptera were investigated for metal
content at the working mandible edge. Multiple samples for each species were assessed
with from 3 to 30 worker individuals measured. The exception was Coptotermes
formosanus where only a single worker was available, however this species has been
investigated previously (Yoshimura et al. 2002; 2005). A minimum of 3 EDS
measurements were taken from each sample. Individuals selected for sampling were well
tanned: showed dark head capsules and mandibles.
Soil samples were taken from seven termite collection sites around South East
Queensland (Australia). These were dried at 60° C and sieved to >63µm<2mm and <2 mm.
Samples (1g) were digested using aqua regia following the Australian Standard method (AS
4479.2). The digest solutions were filtered and made up with 1% nitric acid. Analysis of
Mn and Zn were performed by inductively-coupled plasma optical emission spectrometry.
3
Standard reference materials used were River Sediments NCS CRF NCS DC78301 and
GBW08301. All analyses were performed by Queensland Health Scientific Services NATA
registered (ISO 17025 accredited) Inorganic Chemistry laboratory at Coopers Plains, QLD,
4108.
A colony that was known to be devoid of metals at the mandible edge of worker
individuals was selected for study. Thirty Mastotermes darwiniensis (Mastotermitinae)
mandibles were harvested and treated in one of three ways. One set was exposed only to
water for the duration of the experiment, the second set was exposed to 1M aqueous zinc
acetate dehydrate (C
2
H
3
O
2
)
2
Zn: Sigma Aldrich) for 12 hr, and the third set was pretreated
with 1M (4%) aqueous sodium hydroxide (NaOH: Ajax) at 60°C for 1 hr followed by 2 hr
at room temperature before being washed in deionised water and then exposed to the 1M
zinc acetate for 12 hr at room temperature. All samples were then washed in deionised
water to remove external Zn and air-dried. Mandible edges were assessed using X-ray
analysis (EDS) as above.
Using the Hysitron Triboindentor, scratch tests were applied to a set of termite
mandibles comprising three each of Cryptotermes primus (Kalotermitidae), Coptotermes
acinaciformis ((Rhinotermitidae) and M. darwiniensis (Mastotermitidae). Samples were
embedded in Daystar resin, polymerized at room temperature then polished flat into the
longitudinal plane of the tip region. All tests were at room temperature and the samples
were firmly fixed to the holder. A conical diamond probe tip with a tip radius of 1 µm and a
cone angle of 90° was applied with a normal load of 300 µN and a scratch length of 10 µm.
Penetration into the sample varied from 150 to 200 nm and SEM observation on samples
after testing showed no pileup and no gouging. Mass percentage of elements (e.g. Zn and
Mn) was confirmed after scratch testing using X-ray analysis (as above). Scratch
morphology was investigated using secondary electron imaging on a JEOL 6300 field
emission SEM.
Results
Metal content in the mandible edges is consistent within certain groups (Table 1). A
summary of the metal content by subfamily and life type is presented in Fig. 1. Major Zn
occurrence appears to be present across the whole Kaloterminitidae (drywood termites). It
should be noted that representatives of all major groups in the drywood termites have been
assessed (Fig. 2).
Soil sample data were collected from seven sites where Rhinotermitidae, Termitidae
and Kalotermitidae were taken. All sites showed presence of both Mn and Zn but
concentrations varied from 17 - 1988 mg/kg (dry weight) for Mn and 5 - 211 mg/kg (dry
weight) for Zn. However, there was no correlation between metals in the mandibles of the
termites and soil. The Pearson’s correlation coefficients are as follows: for Mn in the
mandible against Mn in the soil r
9
= -0.078, P = 0.841; for Zn in the mandible against Zn in
the soil r
9
= -0.040, P = 0.920.
Representative X-ray analysis (EDS) spectra are presented in Fig. 3. The halogen
chlorine (Cl) is always associated with the presence of Zn (e.g. Fig. 3b: Cryptotermes
brevis) whereas, when Mn is present alone, while silicon (Si) is still seen, Cl is not always
present (e.g. Fig. 3c: Nasutitermes magnus). The mandibles of M. darwiniensis investigated
for Zn uptake in vitro did not contain Mn or Zn (Fig. 3d), however some regions showed
minor quantities of sulphur (S). Representatives from other colonies of M. darwiniensis
showed trace Mn (Table 1, Fig. 1). The grinding plate, cutting edge and tooth/teeth
characteristic of wood-feeding termites are visible in the mandible of M. darwiniensis (Fig.
4
4a). Sections through the apical tip region and grinding plate were assessed for Zn uptake
using EDS and backscattered electron microscopy (Figs 3d-g, 4b-d). In drywood termites
the cuticle in these regions were seen to contain multiple small pore canals as well as larger
pore canals (see Cribb et al. 2007). These large canals are also evident in transverse regions
of M. darwiniensis mandible edges (Fig. 4 b-d).
Mandibles treated with aqueous zinc acetate, or immersed only in water, did not take
up Zn within the cuticle (see Figs. 3d, 4b, 4d). Only mandibles pretreated with 4% NaOH
solution before exposure to aqueous zinc acetate solution (an uncomplexed form of zinc)
were seen to bind Zn (Figs. 3e, 4c), and the enrichment was not homogeneous. X-ray
analysis (EDS) demonstrated that major Zn uptake occurred principally in the mandible
edge with minor uptake throughout the adjacent insect cuticle (contrast Figs. 3e and 3f with
3g).
The coefficient of friction was assessed for termite mandibles using a scratch test.
This coefficient is defined as the ratio of tangential to normal force (F
t
/F
n
). Values varied as
follows: Cr. primus (Kalotermitidae) averaged 0.25 with a range of 0.24-0.27, M.
darwiniensis (Mastotermitidae) averaged 0.27 with a range of 0.25-0.28 and C.
acinaciformis (Rhinotermitidae) averaged 0.29 with a range of 0.25-0.30. The data sets
from each of the three species tested showed overlap but the lowest friction measurements
were observed from mandibles of Cr. primus. Microanalysis via X-ray analysis confirmed
that the Cr. primus mandibles contained Zn at major levels, the C. acinaciformis mandibles
contained Mn at minor levels and the M. darwiniensis mandibles contained no Zn or Mn.
Table 1. Termite taxa investigated for zinc and manganese incorporation
a
into the mandible
edge of the worker cast using Energy Dispersive X-ray analysis.
Subfamily
Genus and species
Mn
Zn
Location and collection data
Mastotermitidae
Mastotermes darwiniensis
M. darwiniensis
M. darwiniensis
trace
nil
nil
nil
nil
nil
50km NW Townsville,
Queensland, Australia, Feb. 2006.
Mt Carbine, Queensland, Australia,
1966.
Charters Towers, Queensland,
Australia, March 2007
Hodotermitidae
Hodotermes mossambicus
nil
nil
De Aar, Phillipstown. South
Africa, 1961.
Microhodotermes viator
nil
nil
Klaarstroom, Prince Albert. South
Africa, 1963.
Termopsidae
Porotermes adamsoni
P. adamsoni
trace-minor
trace
nil
trace-
minor
Goomburra (Main Range, west of
Aratula), Queensland, Australia,
Feb 2006.
Lamington National Park,
Queensland, Australia, 1958.
Stolotermes victoriensis
nil
trace-
minor
Mt Glorious, Queensland,
Australia, 1963.
Kalotermitidae
Ceratokalotermes spoliator
nil
major
Cornubia, Brisbane, Queensland,
Australia, March 2006.
Cryptotermes primus
trace
major
St Lucia, Brisbane, Queensland,
Australia, Feb. 2006.
Mt Nebo, Queensland, Australia,
March 2006.
5
Cryptotermes brevis
minor
major
Australia, 2006.
Cryptotermes domesticus
trace
“high
concentrat
ion”
Published data (Ohmura et al.
2007)
Cryptotermes queenslandis
minor
major
Tansey, Queensland, Australia,
1967.
Glyptotermes brevicornis
nil
major
St Lucia, Brisbane, Queensland,
Australia, April 2006.
Neotermes insularis
nil
major
Unknown locality, 1953.
N. insularis
nil
major
Babinda, North Queensland,
Australia, 1966.
Incisitermes minor
trace
“high
concentrat
ion”
Published data (Ohmura et al.
2007)
Serritermitidae
Serritermes serrifer
minor
nil
Brazil, 2006.
Glossotermes sulcatus
minor
nil
Brazil, 2006.
Rhinotermitidae
Coptotermes acinaciformis
C. acinaciformis
C. acinaciformis
C. acinaciformis
C. acinaciformis
C. acinaciformis
nil-minor
minor
minor
minor
nil
trace-minor
nil
nil
nil
minor
minor
nil
Narangbar, Queensland, Australia,
2006.
Mt Nebo, Queensland, Australia,
March 2006. Samford Valley,
Queensland, Australia, 2006.
Bundaberg, Queensland, Australia,
1937.
Brisbane, Queensland, Australia,
1937.
Rollingstone, Queensland,
Australia, March 2007 (two nests
evaluated)
Coptotermes frenchi
minor
nil
Queensland, Australia
Coptotermes formosanus
trace
nil
Hollindale, Florida, USA, 1985.
C. formosanus
trace
-
Published data (Yoshimura et al.
2002; 2005).
Heterotermes paradoxus
minor
nil
Forest Lake, Brisbane,
Queensland, Australia, 2006.
Heterotermes ferox
trace
nil
Constitution Hill, Windsor,
Queensland, Australia, Aug. 2005.
Schedorhinotermes
intermedius
minor
nil
Acacia Ridge, Brisbane,
Queensland, Australia, 2006.
Schedorhinotermes seclusus
trace
nil
Acacia Ridge, Brisbane,
Queensland, Australia, 2006.
Reticulitermes hesperus
minor
nil
USA, 2006.
Termitidae
Termitinae
Amitermes laurensis
nil
minor
Chewko, North Queensland,
Australia, 1954.
Amitermes vitiosus
nil
minor
Guluguba, Queensland, Australia,
1959.
Amitermes meridionalis
minor
nil
Litchfield Park, Northern
Territory, Australia, Nov. 2006.
Incolitermes pumilus
nil
minor
Coffs Harbour, NSW,
Australia,1963.
Cristatitermes froggatti
nil
minor
Julatten, North Queensland,
Australia, 1965.
Ephelotermes cheeli
nil
minor
Iron Range, Cape York Peninsular,
North Queensland, Australia, 1971.
Ephelotermes taylori
nil
nil
Litchfield Park, Northern
6
Territory, Australia, Nov. 2006.
Associated with Amitermes
meridionalis nest.
Protocapritermes krisiformis
nil
minor
Lamington National Park,
Queensland, Australia, 1966.
Macrotermitinae
Microcerotermes turneri
minor
trace
Forest Lake, Brisbane,
Queensland, Australia, 2006.
Nasutitermitinae
Nasutitermes triodiae
nil
minor
Mareeba, Paradise Rd, Australia,
1959.
Nasutitermes exitiosus
nil
minor
Australia, 1937.
Nasutitermes pluvialis
nil
minor
Brisbane, Queensland, Australia,
1941.
Nasutitermes graveolus
nil
minor
Mt Steel, 1911.
Nasutitermes magnus
trace -minor
nil
Forest Lake, Brisbane,
Queensland, Australia, 2006.
Nasutitermes fumigatus
trace
nil
Mt Nebo, Brisbane, Queensland,
Australia, 2006.
Cornitermes cumulans
trace
-
Published data (Fawke et al. 1997)
a
Mass reported qualitatively as trace (<0.5 mass % dry weight), minor (a few percent) or
major (>5 - ≥20 % mass weight) (following Newbury 1991).
Fig. 1 Majority rule consensus phylogenetic tree to subfamily level for termites (following
Eggleston 2001; Lo et al. 2007) with metal profile and life history data for workers
(following Abe 1987; Eggleston and Tayashu 2001; unpublished collection data). Metal
concentration for Zn and Mn is mass, reported qualitatively as trace (<0.5 mass % dry
weight), minor (a few percent) or major (>5 to ≥20 % mass weight).
7
Fig. 2 Australian lineage of the Kalotermitidae or drywood termites (following Thompson
et al. 2000) indicating the branches investigated for metal incorporation (bolded). All
branches studied showed major Zn occurrence in the mandible edges of workers. Data on
the metal incorporation is provided in Table 1.
Fig. 3 Elemental composition as representative spectra from energy dispersive X-ray
analysis of termite mandibles. a Daystar polyester resin mounting-medium (control). b
8
Spectrum demonstrating Zn (major quantity) in Cryptotermes brevis; c Mn (minor) in
Nasutitermes magnus; d no metals in this sample from Mastotermes darwiniensis; e M.
darwiniensis mandible tip treated with NaOH prior to zinc acetate (see Fig. 4c for image) f
M. darwiniensis mandible grinding plate treated with NaOH prior to zinc acetate g M.
darwiniensis mandible below edge, treated with NaOH prior to zinc acetate.
Fig. 4 Mandibles of Mastotermes darwiniensis. a Scanning electron micrograph showing
the posterior end a grinding plate (G), anterior cutting edges (C) and tooth (T). Bar: 100
µm. b-d Transverse sections through mandible tips and grinding plate. Images obtained
using backscattered electron imaging: atomic number is correlated with contrast (see J.
Goldstein et al. 2003). Arrows indicate large pore canals. b Mandible tip: treated with zinc
acetate alone. Bar: 10 µm. c Mandible tip: treated with sodium hydroxide prior to zinc
acetate. Metal-enriched areas are whiter than surrounding regions. Bar: 10 µm. d Grinding
plate: treated with zinc acetate alone. Bar: 20 µm.
Discussion
Incorporation of Zn and Mn occurs throughout phylogenies within the Arthropoda
suggesting its advent predates the divergence of the Insecta (Schofield et al. 2003).
Termites (Isoptera) are ancestrally related to wood-feeding cockroaches and have existed
9
for over 100 million years (Pearce 1997; Abe and Bignell 2000; Eggleston and Tayashu
2001; Inward et al. 2007, Lo et al. 2007). However, while we have shown that termite
mandibles generally contain metals, cockroaches that have been investigated do not
incorporate Zn or Mn into their mandibles (Hillerton and Vincent 1982). Within the
Isoptera, our study shows metal incorporation maps onto phylogenetic position and life
type. Mastotermitidae and Hodotermitidae contain no Zn and little to no Mn (Fig. 2)
suggesting that these states are ancestral for termites: the two taxa are considered basal
clades (Pearce 1997). The remaining five subfamilies, in addition to accumulating low
levels of Mn, all show the capacity for acquisition of Zn in the mandible edge, except for
the Serritermitidae (found only in Brazil). The feeding habit of the Serritermitidae is not
known and may be atypical, involving predation and scavenging (Emerson and Krishna
1975). These unusual termites have reduced grinding features on the mandibles unlike
those seen in wood-feeders (Emerson and Krishna 1975). Here the absence of Zn may
represent a secondary loss and is possibly related to adaptation to a different life history;
alternatively the data also support multiple acquisitions for Zn in the phylogenetic tree
since some members of the sister groups, Rhinotermitidae and Termitidae also show an
absence of Zn in the mandibles.
There are examples from other taxa suggesting similar loss of metal acquisition. In
the Hymenoptera mandibles from wasps in the Vespidae and Pelecinidae lack Zn (Quicke
et al. 1998). In the Coleoptera, lack of metals in Carabid mandibles may be a secondary
loss (Hillerton et al. 1984). However dietary deficiency cannot be ruled out as a cause for
absence of metals (Morgan et al. 2003). To address this possibility our study considered
dietary sufficiency. We found colonies within the Rhinotermitidae (Coptotermes
acinaciformis) that lacked Zn but were co-located with a colony from the Kalotermitidae
(Cryptotermes primus) which contained major amounts of Zn in the mandible edges. Both
species were feeding on the same wood and presumably had access to equivalent dietary
Zn, which for the kalotermitids was a sufficient food source on its own for Zn enrichment.
There are some data that indicate concentration of available Mn in the environment
may influence uptake in insects (Morgan et al. 2003). We sampled and tested soil from
termite collection sites to determine whether soil concentration might be linked to metals
present in mandibles. Presumably vegetation supported by the soil would have had access
to the mineral profile but the plant material was not evaluated. Sites tested for a range of
termites collected in South East Queensland (Australia) all showed presence of Mn and Zn
but concentrations varied and there was no correlation between soil concentration and
qualitative concentration in the mandibles. This suggests that metal uptake is not linked to
environmental levels, beyond the need for the metal to be present at some concentration.
Interestingly, within a few of the species sampled the metal profile was seen to vary.
For example, some collections of C. acinaciformis showed minor levels of Zn in their
mandibles whereas others lacked the metal (Table 1). However, evidence based on the
mitochondrial cytochrome oxidase (COII) gene suggests this species probably represents a
complex (Lo et al. 2006). So, such diversity within our sample set may explain the
difference in the observed metal accumulation. The disparity highlights possible
physiological differences. It is worth noting though, that the species complex is expected to
feed in a similar fashion so those members accumulating Zn at a low level would not be
likely to represent a change in feeding strategy.
Since high Zn levels have been linked with enhanced hardness, acquisition within the
Insecta suggests adaptation to extreme environments where improvements in ‘tools’ is an
advantage. An elegant study by Schofield et al. (2002) using leaf-cutter ants supports this
hypothesis. The authors show that ants only cut leaves when the mandible edges increase in
10
hardness, which is accompanied by the incorporation of Zn. Other examples also exist.
Some beetles, like Sitophilus granarius, the granary beetle, have Mn in adult mandibles but
not in the larval mandible (Morgan et al. 2003). In this species the adult penetrates the seed
for the larva, laying the egg within the grain (Morgan et al. 2003). Ichneumonid wasps like
Megarhyssa spp, which contain Mn in the ovipositor, take an hour to drill an oviposition
hole and lay one egg (Quicke et al. 1998). On average such a wasp spends 20 hours actively
drilling. The first instar of the bug Leptoglossus occidentalis which feeds on foliage, lack
Mn but the later instar and adult contain Mn in the rostrum and are seed feeders (Fontaine
et al. 1991). It seems likely that the presence of major quantities of Zn in the drywood
termites might also be linked to a life-history or survival strategy found in this group.
Most termites require contact with water but the Kalotermitidae are referred to as the
drywood termites because they consume sound wood in the absence of free water. They
live in small family nests (a few hundred individuals) within single pieces of wood often
well above the soil. Single-piece nesters, as this life type has been termed (Abe 1987) are
small, confined colonies restricted in foraging choice since they do not leave the nest region
to visit the soil or to scavenge on a range of cellulose sources. The most likely advantage of
the specialized mandibles therefore relates to water use. Workers from other termite groups
have access to free water and can use this. For example Reticulitermes santonensis
(Rhinotermitidae) have been shown to regurgitate water onto substrates from their water
sacs (labial gland reservoirs) (Grube and Rudolph 1999). They are therefore able to bring
water to dry wood surfaces under harvest. These glands also produce a general signal in
termites that results in gnawing, feeding and aggregations at the labeled food site (Reinhard
and Kaib 2001). In contrast, the Kalotermitidae are water limited since they only obtain
water metabolically (Pearce 1997). We hypothesise that whereas most termites can feed on
water-softened food and hence have no need for mandibles with increased hardness,
drywood termites with no access to free water instead harden their mandibles. This may
have resulted in two different strategies in the Isoptera to cope with a difficult food source:
soften it with water, and if this cannot be achieved, develop better tools.
However, groups other than the drywood termites also show uptake or accumulation
of Zn, for example some genera within the higher termites or Termitidae, but these
incorporate Zn to a lesser extent. This does indicate that a mechanism for Zn incorporation
exists outside the Kalotermitidae. At this stage no evaluation has been made of mandible
hardness for minor concentrations of Zn. There can only be an assumption on the basis of
studies from other arthropods that there is a correlation between hardness and
concentration. The presence of a mechanism for Zn incorporation outside the
Kalotermitidae allows that it could be acted upon by selective pressure in these organisms
to produce higher concentrations (as seen in the kalotermitids) and presumably harder
mandibles over evolutionary time.
But are termites that show no Zn uptake in possession of this mechanism also? The
bonding characteristics for Zn in cuticle are poorly understood at present (Schofield 2005)
with evidence from marine worms pointing towards possible involvement of proteins,
specifically the metal-binding imidazole side chain of histidine (Lichtenegger et al. 2003,
Broomell et al. 2007). The microstructure is better documented. Schofield et al. (2003)
noted that arthropod cuticle with high densities of pore canals is associated with metal
uptake and Compère et al. (1993) demonstrated experimentally that pore canals are the
means by which biomineralisation precursors are transferred to the cuticle, in this case
calcium and magnesium ions, in crabs. So presence of these canals can be taken as an
indicator of cuticle that is associated with increasing metal mass. Cribb et al. (2007) noted
pore canals in the mandible edges of Kalotermitidae. Both small and large canals were
11
identified. In the current study the mandible edges of Mastotermes darwiniensis appear
similar. But this is not surprising since trace amounts of Mn have been found in the edges
of some mandibles from this species. The canals are likely to be the physical transport
system for translocation of metals. Active binding sites must also be present for
incorporation to occur.
To explore further, we undertook an experiment aimed at testing whether mandibles
of M. darwiniensis could take up Zn under in vitro conditions. The specimens used were
initially devoid of metals. Results demonstrated that uptake occurs, but only if the cuticle is
pretreated with NaOH. Since there was no obvious degradation in the microstructure at the
mandible edge, presumably the treatment did not extract structural elements. Cuticle is
comprised of chitin chains and surrounding proteins, however a harsher protocol (5%
NaOH at 100°C) than that used would be needed to remove those proteins attached directly
to the chitin fibres (Vincent and Wegst 2004) or cause chitin to undergo significant
deacetylation (45% NaOH for 30 min at 15 psi and 121°C) (No et al. 2000). It is therefore
likely that proteins were principally affected.
Uptake of Zn only after cuticle pretreatment with NaOH is likely to occur because
any protein present will be in anionic form at the pH of 4% NaOH solution (1 M and
pH>12) and likely to exceed the isoelectric point of the protein (Sloane and York 1969).
Zinc in acetate solution is an uncomplexed form and will therefore bind to any protein
activated in anionic form.
What is interesting is that Zn enrichment occurred in the cuticle in a similar pattern to
enrichment in the drywood termites (as presented in Cribb et al. 2007). That is, rather than
occurring homogeneously throughout the cuticle, the major enrichment occurred only at the
edge. This is despite the edge being heavily tanned and therefore less prone to cleaving of
molecular bonds. The data suggest a specific biochemistry, presumably associated with the
protein complex, suited to metal-bonding in this area. Schofield (2001) has argued that
evidence from arthropods indicates there are two distinct processes, one associated with Zn
uptake and a second with Mn and Ca uptake, so an ability to bind one does not necessarily
imply an ability to bind the other. Whatever the case, currently only the drywood termites
show the capacity to take up major quantities of Zn into the mandible edge in live
individuals, and the presence of this metal in major quantities has been linked to possession
of harder mandibles (Cribb et al. 2007).
Cribb et al. (2007) demonstrated that mandibles of termites containing Zn are 20%
harder than those containing Mn or no metal. This raised the prospect that mandibles
containing Zn may undergo less wear. We applied a scratch test to a number of termite
mandibles principally to investigate scratch resistance. Such tests mimic interaction of the
mandibles with hard materials in their environment such as during feeding and nest
building activities. Wei and Bhushan (2006) highlight that little work has been done in the
area of scratch tests for biomaterials and canvas the complexity of comparing materials
with differing hardness values. However, it should be noted that in the scratch test, unlike
some samples from the work of Wei and Bhushan (2006) which caused difficulties in
comparison, there was no evidence of the mandible tissues having been gouged or cut into
by the tip during scratching - in fact there was no evidence of the scratch after the force was
removed. Considering termite mandibles containing Zn are harder than those with low
levels of Mn or no metals (Cribb et al. 2007), it is not surprising that the smallest values
(down to 0.24) for coefficient of friction were found in this Zn-containing group.
It must not be forgotten that hydration level of materials is also significant. Schöberl
and Jäger (2006) have shown that more material is removed by wear on wet cuticle than dry
cuticle (of the marine worm, Nereis) despite being less hard. This cuticle also has a lower
12
friction as measured by lateral force microscopy (a qualitative assessment) than dry cuticle.
Although a larger data set would be beneficial, including one that assesses the use of a
range of normal force application and hydration states, the current data obtained for termite
mandibles can be interpreted as showing that dry Zn-containing mandibles are likely to be
more scratch resistant than mandibles lacking Zn, hence possibly more resistant to
environmental degradation. In contrast lack of metals or presence of low mass % of Mn
does not appear to provide an advantage in terms of coefficient of friction. We must
continue to look elsewhere for an explanation for why Mn is incorporated into cuticle at
these low levels.
Acknowlegements We gratefully acknowledge supply of termite samples from Prof.
Reginaldo Constantino, Universidade de Brasília, Brazil; Mrs Vivienne Uys, Plant
Protection Research Institute, Queenswood, South Africa and Dr Gerhard Prinsloo,
Agricultural Research Council-Plant Protection Research Institute, Pretoria, South Africa;
Dr Michael K. Rust, Department of Entomology, UCR, Riverside, USA; Dr B. Peters,
Queensland Department of Primary Industries and Fisheries; Mr Greg Daniels, The
University of Queensland Insect Collection, Brisbane, Australia; and Mrs Rachel Hancock
Narangbar, Queensland, Australia. We thank Mr. Yueqin Wu, University of Queensland,
for his experimental assistance in scratching tests and Dr Michael Lenz, CSIRO
Entomology, Canberra, and Dr David Merritt and Dr Lyn Cook, University of Queensland
for their helpful comments on the manuscript when in preparation. The experiments comply
with the current laws of the country in which they were performed.
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