Gabriel A. Miller, School of Biological Sciences, The University of Sydney, Heydon-Laurence Building A08, NSW 2006, Australia. E-mail: firstname.lastname@example.org
1. The density-dependent prophylaxis hypothesis predicts that individuals in high-density populations will invest more resources in immune defence than individuals at lower densities.
2. However, recent work suggests that this prediction may not apply to all situations; solitarious species may paradoxically have higher scores than crowded counterparts in certain immune assays.
3. To investigate the relationship between a key immune parameter and field population densities, the total haemocyte counts (THCs) of Australian plague locusts (Chortoicetes terminifera) from three population densities in Western Australia were compared.
4. THCs were negatively correlated with field population densities, and locusts removed from a marching band and kept in isolation had increased THCs relative to group-housed controls.
5. These results demonstrate that immune investment can inversely relate to population density in field conditions.
6. We suggest that isolated locusts increase their haemocyte densities relative to crowded conspecifics in response to potentially greater exposure to parasitoids and nematodes.
High population densities have been linked to increased rates of parasitism (Sherman et al., 1988; Schmid-Hempel, 1998). Accordingly, the density-dependent prophylaxis hypothesis predicts that organisms living in high population densities will invest more in immune defence (Wilson & Reeson, 1998). However, this prediction does not hold for all group sizes or when pathogen spread does not depend upon host contact; indeed, contingent upon such parameters, immune investment may be unrelated, inversely related or non-linearly related to population density (Watve & Jog, 1997; Naug & Camazine, 2002; Wilson et al., 2003). Little is known of host immune defence in field populations where pathogen prevalence and host contact rates are not well characterised.
The complexity of interactions between population density and immune investment is exemplified by experiments in which solitary-living Lepidoptera were found to have boosted immune parameters relative to phylogenetically-paired, identically-reared gregarious species (Wilson et al., 2003). This observation is opposite to that predicted by basic density-dependent prophylaxis. In each of the six species pairs, THCs were significantly higher in species adapted to low population densities, reiterating earlier theoretical work (e.g. Naug & Camazine, 2002) suggesting that predictions of the density-dependent prophylaxis hypothesis may benefit by inclusion of additional information about relevant host–parasite interactions.
Locusts are a model system for understanding relationships between immunity and population density due in part to the species' density-dependent phenotypic plasticity (reviewed by Pener & Simpson, 2009). Such plasticity extends to immune function: pathogen resistance varies as a function of locust population density in laboratory conditions (e.g. Wilson et al., 2002; Miller et al., 2009a). However, little is known about the influence of population density upon locust immune parameters in wild populations.
The aim of the present study was to investigate the density dependence of locust immunity in field conditions. Immune defence can be measured either by direct immune challenges or by assessment of various immune parameters. Presentation of insects with a pathogen challenge provides a functional assessment of resistance, encompassing behavioural and other external mechanisms in addition to cellular and humoral immunity (Hughes et al., 2002; Traniello et al., 2002). Assays of haemolymph properties serve in parallel with pathogen challenges to define host resistance (e.g. Wilson et al., 2002). Total haemocyte count (THC), the density of cells in the haemolymph, is central to a host's capacity to defend against metazoan parasites (Kraaijeveld et al., 2001; Wilson et al., 2003).
To assess the relationship between an immune parameter and population density, we measured THCs of individuals within Australian plague locust (Chortoicetes terminifera) populations of varying densities. In addition, individuals from a high-density marching band were maintained in isolation to determine whether THCs change in response to decreased population densities.
Materials and methods
Field sites and conditions
Fifth-instar Australian plague locusts (C. terminifera) were obtained in field conditions from three sites (roughly forming an isosceles triangle with dimensions of 40 and 15 km; see Table 1) near Ravensthorpe, Western Australia from 19–23 November 2007. All sites were approximately 350 m above sea level and contained similar vegetation. The density of grasshoppers at each field site (Table 1) was determined by averaging measurements from 20 replicate quadrats, each with area 0.38 m2. All populations were present in a region with extensive locust swarm activity and are likely to have been members of gregarious populations within weeks prior to experimentation; the behavioural phenotype of various populations is the subject of ongoing analyses. Temperatures at 15.00 hours during sample days were 23°C ± 2° SE (Australian Bureau of Meteorology recording station, Ravensthorpe, Washington).
Table 1. Field site locations and locust density measurements.
*For each location, average of 20 replicate quadrat measurements.
S 33° 21.794′ E 120° 10.994′
S 33° 21.794′ E 120° 10.994′
S 33° 21.794′ E 120° 10.994′
No group movement
S 33° 30.279′ E 119° 48.662′
No group movement
S 33° 14.989′ E 120° 08.542′
Insect capture and group/solitary housing conditions
On Day 0, 97 locusts were captured from a marching band at Site 1 using sweep nets and either immediately sacrificed for THC measurements (n = 21) or placed into group (n = 43) or solitary (n = 33) housings (as described below). The following day (Day 1), 30 group-housed and 19 solitary-housed insects were sacrificed for THC assessment. On Day 2, the remaining 13 group-housed and 14 solitary-housed insects were similarly sacrificed. Group-housed locusts were kept in the shade in 30 cm3 wire-framed nylon mesh enclosures each with approximately 100 conspecific fifth-instars. Solitary-housed locusts were placed in the shade in visually-isolated inverted cylindrical plastic pots (15 cm high × 6 cm diameter) fitted with two large nylon mesh windows to aid ventilation. All insects were given fresh lettuce daily. Insects from sites 2 and 3 (n = 6 and n = 13, respectively) were captured with sweep nets and immediately bled for THC measurements.
Measurement of total haemocyte counts
Insects were singly removed from housings (alternating group and solitary housed) and bled by hind femur removal to yield 5–10 µl of haemolymph each. Samples were transferred with a micropipette beneath the coverslips of improved Neubauer haemocytometers (Scientific Laboratory Supplies, Nottingham, U.K.), and haemocyte densities were obtained by averaging counts in five non-adjacent squares for each slide (quantified manually by compound microscope).
All data were analysed in SPSS 14 (SPSS Inc., Chicago, Illinois). To meet assumptions of parametric statistics, THCs were log-transformed. For visualisation, values were reverse transformed and plotted with asymmetric error bars. Pearson's coefficients were used to assess correlations between haemocyte count and population density; the latter was assumed to be the major variant between sites. Two-sample t-tests were used to compare group- and solitary-housed THCs on day 1 and (separately) day 2. Finally, a linear contrast anova was employed to check whether THCs increased in a dose-dependent manner with the duration of solitary housing. Significance values were adjusted for repeated contrasts using Bonferroni corrections.
Chortoicetes terminifera total haemoctye counts correlated with population densities (Fig. 1; Pearson's coefficient = −0.45, P = 0.004, N = 40). Furthermore, solitary housing induced a dose-dependent increase in THCs (Fig. 2; one-way anova linear contrast for Days 0–2, P = 0.023). Two days of solitary housing were sufficient to increase THC relative to controls [Fig. 2; two-sample t(25), t = 2.535, Bonferroni-corrected P = 0.036]. Solitary-housed insects also had greater mean THCs than controls on Day 1 (Fig. 2), but these differences were not statistically significant [two-sample t(47), t = 1.685, Bonferroni-corrected P = 0.198].
Total haemocyte count related to population density in a manner opposite to that expected by positive density-dependent prophylaxis. Low C. terminifera population densities were correlated with increased THCs (Fig. 1). Furthermore, over a period of 2 days, solitary housing in a field setting dose-dependently increased THCs relative to group-housed controls (Fig. 2). These results strengthen the assertion that increased population density does not imply uniformly increased immune investment. The negative density dependence of THC represents data from three field sites assessed on different days, allowing for alternative explanations of THC differences attributable to temperature or other variable phenomena. However, given that the same density-dependent effect was observed in a location- and time-controlled experiment (Fig. 2), population density seems a plausible explanation for site-related THC variation (Fig. 1).
Pathogen behaviour may determine aspects of immune investment in concert with host population density. In a meta-analysis, Côté and Poulin (1995) determined that pathogens which spread by direct contact were likely to induce positive density-dependent prophylaxis in host populations. Furthermore, these authors demonstrated that mobile pathogens, which actively locate hosts and do not require direct contact for propagation, might invoke negative density-dependent prophylaxis. In such cases, one could imagine a protective effect of operating in groups similar to that which offers protection against predators (Mooring & Hart, 1992). In such systems, decreased encounter rates between gregarious (high-density) populations and pathogens or predators may compensate for increased intra-group contact rates (Watve & Jog, 1997; Wilson et al., 2003; Miller et al., 2009b).
The importance of the distance between host groups in host–pathogen interactions is appreciated in models incorporating ‘percolation theory’ (Reynolds et al., 2009). In this framework, pathogens move through host landscapes similarly to the way fires move through forested areas. Forests with isolated clumps of trees, corresponding to populations of gregarious insects, may be less susceptible to fire propagation (or mobile pathogen attack) compared with forests with homogenous tree distributions (Wilson, 2009).
Immune assays such as THC measurement are limited in interpretational scope, and drawing general conclusions from such studies about immunocompetence can be problematic. As pointed out by Owens and Wilson (1999), a unitary concept of immunocompetence is difficult to establish. Immune parameters often independently vary (e.g. Wilson et al., 2002; Cotter et al., 2004; Povey et al., 2009), and therefore may not be reliable indicators of a host's susceptibility to attack by particular pathogens. For example, in a study conducted on adult desert locusts (S. gregaria), Wilson et al. (2002) found no significant differences in encapsulation response between solitarious and gregarious locusts, yet gregarious locusts demonstrated higher survival to a fungal challenge.
As the maintenance and upregulation of immune responses are costly, the expression of immune function is expected to depend upon the condition of the individual (Blanco et al., 2001; Cotter et al., 2004). Host species are therefore likely to vary in their propensity to manifest THC changes in response to a disease threat. Studies in African cotton leafworms (Spodoptera littoralis) and desert locusts (Schistocerca gregaria) found marginal, but not significant, increases in THC with population density (Wilson et al., 2002; Cotter et al., 2004). However, the present results (Figs 1 and 2), and those of Wilson et al. (2003), are opposite to this trend, suggesting that different species may have encountered varying pathogen strategies (e.g. mobile or host contact-dependent spreading) through evolutionary history.
Increased THC in solitary-housed C. terminifera locusts suggests that locusts may be evolutionarily prepared for potential increases in the exposure of solitary insects to mobile pathogens such as nematodes or parasitoids. Indeed, mermithid nematodes, in addition to nemestrinid, scelionid, and sarcophagid parasitoids, are known to impact C. terminifera populations (Baker & Pigott, 1995). However, we cannot rule out the possibility that forces unrelated to immune function triggered observed THC increases, and that boosted immunocompetence (if it occurred) was epiphenomenal. For example, the smaller size of solitarious locusts relative to gregarious counterparts (Uvarov, 1966) may result in lower solitarious total haemolymph volume, thereby increasing haemocyte density through a concentration effect. This explanation cannot account for the magnitude (nearly 50%) of THC increases after just 2 days of solitary rearing, however, nor is it known whether this size difference applies in Chortoicetes.
Regardless of the speed and mechanisms by which THCs change, increased haemocyte densities clearly promote host defence (recently reviewed by Strand, 2008). Accordingly, haemocytes seem to be a major target of pathogen-mediated immunosuppressive processes (Combes et al., 2001). Further investigation of immune parameters in field conditions will provide a broader understanding of the role of pathogens in group formation and the relations between host immunocompetence and population density.
K. Berthier, J. Buhl, M. Chapuis, and J. Popple provided invaluable assistance with fieldwork. We also thank two anonymous reviewers for very useful commentary. G.A.M. was funded by an Oxford Clarendon Award and a grant from the Orthopterists' Society. All experiments were conducted in accordance with Australian law.