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Currentimmunitymarkersininsectecologicalimmunology:assumed
tradeoffsandmethodologicalissues
M.MorenoGarca,A.CrdobaAguilar,R.CondandH.LanzMendoza
BulletinofEntomologicalResearch/FirstViewArticle/August2012,pp113
DOI:10.1017/S000748531200048X,Publishedonline:
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Introduction
Ecological immunology is a rapidly expanding, young
discipline (Rolff & Reynolds, 2009). Within this field, immune
response is seen as a set of traits that involve costs (Sheldon
& Verhulst, 1996). Resources must be allocated between an
organisms maintenance and proper functioning and the
expression of other traits linked with survival and reproduction, resulting in ecological trade-offs among trait types. At the
moment, there is growing empirical evidence of trade-offs
associated between life-history traits (size at birth, growth and
mortality rates, size and age at maturity, longevity and survival, clutch size and reproductive effort: reviewed in Stearns,
1992) and immune defense (e.g. Boots & Begon, 1993;
Kraaijeveld & Godfray, 1997; Fellowes et al., 1998; Moret &
Schmid-Hempel, 2000; Rigby & Jokela, 2000; Hoang, 2001).
The ecological immunity framework has been applied to a
number of disciplines, including sexual selection (i.e. differential mating and fertilization success: Darwin, 1871) (e.g.
Siva-Jothy, 2000; McKean & Nunney, 2001; Roberts et al., 2004)
and, to a lesser extent, social evolution (e.g. Calleri et al., 2007;
Bocher et al., 2008), predator-prey relationships (e.g. Packer
et al., 2003; Roy & Holt, 2008) and parental investment (e.g.
Hoi-Leitner et al., 2001; Brzek & Konarzewski, 2007). The
influence of ecological immunity has also extended to broader
fields, such as life-history theory (Schmid-Hempel, 2005a),
parasite-host coevolution (e.g. Day et al., 2007; Duffy & SivarsBecker, 2007), conservation biology (e.g. Stevenson, 2006;
M. Moreno-Garca et al.
Cellular responses
Total number of haemocytes (haemocyte density)
Haemocyte counting and/or activity is a straightforward
way of assessing cellular immune ability (e.g. Rantala et al.,
2000; Kraaijeveld et al., 2001; Cotter et al., 2004). Haemocytes
are generally responsible for immune activities, such as
engulfing, surrounding and destroying infectious agents.
After bacteria have penetrated the haemocoel, haemocytes
are the first and most efficient bacteria-clearing mechanism,
(Hillyer et al., 2003). However, there are different haemocyte
types, each playing a different function (recognition, phagocytosis, nodulation, encapsulation or melanization) (see reviews by Lavine & Strand, 2002; Ribeiro & Brehlin, 2006).
Differences in pathogen type (e.g. bacteria, virus, nematodes,
etc.) among individuals, sexes or species must be considered,
for example, for example, the mosquito Aedes aegypti mainly
phagocyte Escherichia coli. In contrast, other types of bacteria
(e.g. Micrococcus luteus) are melanized (Hillyer et al., 2003).
However, the factors eliciting phagocytic vs. melanization
responses against bacteria are independent of Gram type
(Hillyer et al., 2004).
Methodology
For total and viable counts, haemolymph is collected and
incubated in humidity chamber, then counted using an hemocytometer using (phase contrast) microscopy (e.g. Rantala
et al., 2000; Kraaijeveld et al., 2001). For collection and incubation of haemolymph, we suggest the use of cell culture
media, such as RPMI, Schneider or Grace rather than PBS
buffers. This media may allow a better viability of cells.
Trypan blue can be used to differentiate between dead and
viable cells. Haemolymph extraction may be accomplished by
perfusion-bleed; a droplet can be collected from a puncture/
cut on the thorax or by a removed leg. Although this may
appear an easy task, haemocytes can adhere to tissues such as
the fat body. One solution to this problem is to use anticoagulant buffers of low pH (Pech et al., 1994). An accurate
extraction of these cells can also be carried out by injecting a
protease inhibitor blend (e.g. PMSF, TLCK, leupeptine).
However, optimal conditions for obtaining insect haemocytes
cannot be generalized; and, in each procedure of extraction,
the use (or non-use) of anticoagulant must be adjusted.
Phagocytosis
Phagocytic activities require the internalization of the
surface membrane and action of several organelles, including
endosomes and lysosomes, which fuse with the plasma membrane and provide the membrane for phagosome formation
(Desjardins et al., 2005). In mosquitoes, the synthesis of lipids
for the membranes of the phagosome, plasma membrane and
for the production of cytotoxic proteins for the degradation of
phagocytised material decreases their reproductive output
(Ferdig et al., 1993; Hogg & Hurd, 1995; Hillyer et al., 2003). For
these reasons, trade-offs are expected among phagocytic
Table 1. Immune markers used in insect ecological immunity studies (see text for full discussion).
Immune marker
Methodology
Recommendations
Example references
Cuticle
Haemocyte
density
Phagocytosis
Nodule/capsule
formation
Nylon
monofilaments
Nylon/silica
beads
Adamo, 1999
Kumar et al., 2003
Schwartz & Koella,
2004
M. Moreno-Garca et al.
Immune function
Lysozyme activity
Antimicrobial
peptides
Goldsworthy et al.,
2003
Wilson et al., 2003
Jacot et al., 2005
Moret &
Schmid-Hempel,
2000
McKean &
Nunney, 2001
Phenoloxidase
(PO)
* Before inoculation, minimal lethal and/or sub-lethal doses must be determined. Overdoses can promote septic shock, rather than a measurable immunological response.
**Because antimicrobial activity can be due to lysozymes, reactive oxygen species and/or antimicrobial peptides, immune response measurement is commonly referred to as hemolytic, lysozymelike or antibacterial activity.
M. Moreno-Garca et al.
Humoral responses
Phenoloxidase activity
PO is the most widely used immune marker in ecological
immunology studies (e.g. Goldsworthy et al., 2003; Wilson
et al., 2003; Jacot et al., 2005). The PO enzyme is used for different physiological processes (wounding, clotting, cuticle composition, melanotic encapsulation, production of cytotoxic
molecules) and probably spermatheca formation (Ilango,
2005). Due to these different functions, PO is constantly
synthesized; therefore, it could be costly to produce for the
M. Moreno-Garca et al.
problem with assessing PO is that protein load standardization is needed before measurement, using a standard protein
assay (Contreras-Garduo et al., 2007), a practice that is rarely
carried out and without which there may result disparate and
unreliable PO readings. Since, occasionally, real protein load
can be masked if there are differences among individuals (e.g.
bigger size of females than males, inaccurate extract of total
amount of haemolymph). We recommend using the protein
load practice mentioned above. Furthermore, there might be a
rapid degradation of PO if stored for a relatively long time.
This issue can be resolved by using a protease inhibitor
cocktail (e.g. PMSF, Leupeptin). Another problem is that
small-sized insects may not provide PO readings, since PO
quantities are so small. This problem can be solved by using
more than one individual for a single sample to obtain PO
readings. Also, the total PO activity can be estimated using the
enzyme -chymotrypsin to activate all pro-PO present in the
haemolymph (see Bailey & Zuk, 2010).
Nitric oxide
NO quantification is a relatively new immune marker in
ecological immunity studies (see, for example, Moreno-Garca
et al., 2010). NO damages pathogens and is also used as a
signalling molecule. In Drosophila, NO is indispensable for
activation of the immune deficiency (Imd) pathway (Foley &
OFarrel, 2003). This is one of the three pathways (the other
two being Toll and JAK/STAT; see Ferrandon et al., 2007) that
produce transcriptional factors in the fat body, leading to the
synthesis of antimicrobial peptides and other factors that
prevent self tissue damage and control haemocyte proliferation and differentiation (Foley & OFarrell, 2003; Agaisse &
Perrimon, 2004). Another interesting point is the fact that the
NO synthase (NOS) (an enzyme that converts L-Arginine to
L-Citrulline and generates NO) is constitutive and inducible
(Mller, 1997). NO is also a signalling molecule, so a constant
production of NOS is necessary for homeostatic purposes,
while inducible NOS is only synthesized after an immune
challenge (Nappi et al., 2000). It is, therefore, expected that
NO production increases after an immune challenge.
Nevertheless, Krishnana et al. (2006) found that NO production was similar in both non-stimulated and stimulated
haemocytes in lepidopteran larvae. In this particular case, the
authors indicate that NO can be continuously synthesized by
the induced NOS for long periods of time (hours to days). This
means that the effectiveness for killing pathogens with NO
may not only reside on NO concentration but in the duration
of NO synthesis (see Laurent et al., 1996).
NO is produced during the oxidation of L-Arginine
(Mller, 1997). Arginine is an amino acid, which must be
obtained from the diet (Rivero, 2006). Arginine is also important for sperm maturation (Osanai & Chen, 1993), egg
production (Uchida, 1993), long term memory, chemosensory
(antennal lobes, olfaction) and visual information processing
(Mller, 1997). Trade-offs are thus expected among these traits
and the immune response.
Methodology
Basal levels of NO are normally used, which provides an
incomplete picture of host immune ability. A better alternative
is to assess NO production after host injection or ingestion of
pathogens. The standard method to measure NO is to use the
Griess reaction that measures nitrite production, an indirect
Methodology
Lysozyme activity is commonly assayed via the clearance
rate of bacterial suspension using haemolymph. The method
is relatively simple; after the haemolymph is extracted and
mixed with a bacterial solution, a turbidity assay is then performed (e.g. Adamo, 2004). Small absorbance values indicate
high lytic activity. Another way is to add small drops of
haemolymph to bacterial culture, measuring the area of lytic
activity after some time (12 h). The lytic activity appears as
clear circular zones in the culture, which indicates that bacteria
have been cleared. The diameter of the hole is measured and
taken as the indicator of lytic effectiveness.
AMPs have been measured by injection or ingestion
of complete bacteria, fungi, parasites or their fractions (e.g.
LPS, peptidoglycane). AMP activity is assessed in three ways:
(i) measurement of the clearance rate of a bacterial suspension
(via a turbidity assay, similar to the lysozyme protocol described above); (ii) measurement of the rate of bacterial growth
on a plate, after the same bacteria have been injected into the
host and the haemolymph has been extracted after some time;
and (iii) the antibacterial zone assay, via measurement of the
hole area, similar to the lysozyme protocol described above.
A problem with these different assays is that antimicrobial
activity can be due to other molecules and not necessarily
AMPs. Since lysozyme needs a pH 8 to 6.5 for optimal activity,
this can be used to distinguish between other AMP activities
Acknowledgements
We thank Salvador Hernndez-Martnez for providing
assistance with protocols and for helpful suggestions. We
thank Gloria Tavera and Chris Anderson for revising this
paper in English, and providing thoughtful comments. MM-G
was supported by the Posgrado en Ciencias Biomdicas
(CONACYT: grant no. 172947), Instituto de Ecologa, (Universidad Nacional Autnoma de Mxico), and the Centro de
Investigacin Sobre Enfermedades Infecciosas, Instituto
Nacional de Salud Pblica, Mxico. AC-A was supported
by a grant provided by PAPIIT-UNAM (Project No. 211506).
An anonymous reviewer provided corrections to the manuscript.
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