Nephila Plumipes Research Articles

Major ampullate silk gland transcriptomes and fibre proteomes of the golden orb-weavers, Nephila plumipes and Nephila pilipes (Araneae: Nephilidae).

Natural spider silk is one of the world’s toughest proteinaceous materials, yet a truly biomimetic spider silk is elusive even after several decades of intense focus. In this study, Next-Generation Sequencing was utilised to produce transcriptomes of the major ampullate gland of two Australian golden orb-weavers, Nephila plumipes and Nephila pilipes, in order to identify highly expressed predicted proteins that may co-factor in the construction of the final polymer. Furthermore, proteomics was performed by liquid chromatography tandem-mass spectroscopy to analyse the natural solid silk fibre of each species to confirm highly expressed predicted proteins within the silk gland are present in the final silk product. We assembled the silk gland transcriptomes of N. plumipes and N. pilipes into 69,812 and 70,123 contigs, respectively. Gene expression analysis revealed that silk gene sequences were among the most highly expressed and we were able to procure silk sequences from both species in excess of 1,300 amino acids. However, some of the genes with the highest expression values were not able to be identified from our proteomic analysis. Proteome analysis of “reeled” silk fibres of N. plumipes and N. pilipes revealed 29 and 18 proteins, respectively, most of which were identified as silk fibre proteins. This study is the first silk gland specific transcriptome and proteome analysis for these species and will assist in the future development of a biomimetic spider silk.

Plastic material investment in load-bearing silk attachments in spiders

The nature and size of attachments is a fundamental element of animal constructions. Presumably, these adhesive structures are plastically deployed to balance material investment and attachment strength. Here we studied plasticity in dragline anchorages of the golden orb web spider, Nephila plumipes. Specifically, we predict that spiders adjust the size and structure of dragline anchorages with load, i.e. spider mass. Mass was manipulated by attaching lead pieces to the spider’s abdomen resulting in a 50 percent increase in mass. Loaded spiders spun larger but structurally similar thread anchorages than unloaded spiders. Thus, the spinning program that determines the overall anchor structure is highly stereotypic, and flexibility is introduced through varying the anchor size by increasing material investment. Our study showcases substrate attachments as suitable models to investigate the interplay between innate and changeable elements in the economy of building behaviours.

Casual movement speed but not maximal locomotor capacity predicts mate searching success.

Maximal locomotor performance is often used as a proxy for fitness. Maximal speed may be important under high-threat conditions, such as during predator escape. However, animals do not always move at a speed that reflects their maximal physiological capacities when undisturbed. The physiological factors that determine the movement speed chosen by animals, such as minimization of energy use, may be independent from maximal performance. As a result, the casual speed at which individuals move when undisturbed in a given context may better represent an individual's motivation to move. The casual speed may therefore be a better predictor of fitness in natural contexts than maximal performance capacity. We tested the hypothesis that casual movement speed rather than maximal speed predicts fitness in the golden orb-web spider, Nephila plumipes. We measured fitness in two separate contexts, mate-searching success and the positional rank near a female. We show that casual but not maximal locomotor speed predicted both aspects of fitness. Casual speed was linearly related to maximal speed, indicating that casual speed is determined by physiological optimization. Size and metabolic scope were not related to either maximal or chosen speeds, indicating that the supply of ATP does not limit locomotor performance in this species. Overall, our results demonstrate that locomotor performance is related to fitness, but suggest that different types of performance and not necessarily maximal physiological capacities are most relevant for particular ecologically relevant tasks.

Mechanical properties of silk of the Australian golden orb weavers Nephila pilipes and Nephilaplumipes.

ABSTRACTSilks from orb-weaving spiders are exceptionally tough, producing a model polymer for biomimetic fibre development. The mechanical properties of naturally spun silk threads from two species of Australian orb-weavers, Nephila pilipes and Nephila plumipes, were examined here in relation to overall thread diameter, the size and number of fibres within threads, and spider size. N. pilipes, the larger of the two species, had significantly tougher silk with higher strain capacity than its smaller congener, producing threads with average toughness of 150 MJ m−3, despite thread diameter, mean fibre diameter and number of fibres per thread not differing significantly between the two species. Within N. pilipes, smaller silk fibres were produced by larger spiders, yielding tougher threads. In contrast, while spider size was correlated with thread diameter in N. plumipes, there were no clear patterns relating to silk toughness, which suggests that the differences in properties between the silk of the two species arise through differing molecular structure. Our results support previous studies that found that the mechanical properties of silk differ between distantly related spider species, and extends on that work to show that the mechanical and physical properties of silk from more closely related species can also differ remarkably.

Evidence of Decoupling Protein Structure from Spidroin Expression in Spider Dragline Silks.

The exceptional strength and extensibility of spider dragline silk have been thought to be facilitated by two spidroins, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2), under the assumption that protein secondary structures are coupled with the expressed spidroins. We tested this assumption for the dragline silk of three co-existing Australian spiders, Argiope keyserlingi, Latrodectus hasselti and Nephila plumipes. We found that silk amino acid compositions did not differ among spiders collected in May. We extended these analyses temporally and found the amino acid compositions of A. keyserlingi silks to differ when collected in May compared to November, while those of L. hasselti did not. To ascertain whether their secondary structures were decoupled from spidroin expression, we performed solid-state nuclear magnetic resonance spectroscopy (NMR) analysis on the silks of all spiders collected in May. We found the distribution of alanine toward β-sheet and 3,10helix/random coil conformations differed between species, as did their relative crystallinities, with A. keyserlingi having the greatest 3,10helix/random coil composition and N. plumipes the greatest crystallinity. The protein secondary structures correlated with the mechanical properties for each of the silks better than the amino acid compositions. Our findings suggested that a differential distribution of alanine during spinning could decouple secondary structures from spidroin expression ensuring that silks of desirable mechanical properties are consistently produced. Alternative explanations include the possibility that other spidroins were incorporated into some silks.

Persistence and survival of the spider Nephila plumipes in cities: do increased prey resources drive the success of an urban exploiter?

Species that successfully inhabit urban ecosystems are rare, and urbanisation often drives localised extinctions of native species. Nonetheless, some species take advantage of the novel conditions available in cities and increase in abundance. Trends in the abundance and distribution of species in urban areas have received much attention, but the precise elements of urban ecosystems that affect the survival of urban-dwelling species are largely unknown. Animals that successfully exploit urban environments may do so because of increases in the availability of resources or habitats. Here we assess the effects of anthropogenic landscapes and prey abundance on the persistence of an orb-weaving spider, Nephila plumipes. We assessed spider persistence for six months in situ along an urban gradient in Sydney. We then transplanted spiders from a common garden into sites along the gradient, monitored their persistence in the new environment and measured a suite of environmental variables at local and landscape scales. The abundance of prey was closely linked with spider persistence, in both the survey and the transplant experiment, and was positively associated with anthropogenic habitats. The surveyed spiders survived longer when located closer to the coast and transplanted spiders persisted longer in smaller sites with more impervious surfaces and reduced vegetation cover. Our study shows that urbanisation has a strong effect on potential prey abundance and can lead to increased persistence of N. plumipes, demonstrating the broad impacts that habitat disturbance can have on the life history and trophic interactions of city-dwelling animals.

Urbanisation at multiple scales is associated with larger size and higher fecundity of an orb-weaving spider.

Urbanisation modifies landscapes at multiple scales, impacting the local climate and changing the extent and quality of natural habitats. These habitat modifications significantly alter species distributions and can result in increased abundance of select species which are able to exploit novel ecosystems. We examined the effect of urbanisation at local and landscape scales on the body size, lipid reserves and ovary weight of Nephila plumipes, an orb weaving spider commonly found in both urban and natural landscapes. Habitat variables at landscape, local and microhabitat scales were integrated to create a series of indexes that quantified the degree of urbanisation at each site. Spider size was negatively associated with vegetation cover at a landscape scale, and positively associated with hard surfaces and anthropogenic disturbance on a local and microhabitat scale. Ovary weight increased in higher socioeconomic areas and was positively associated with hard surfaces and leaf litter at a local scale. The larger size and increased reproductive capacity of N.plumipes in urban areas show that some species benefit from the habitat changes associated with urbanisation. Our results also highlight the importance of incorporating environmental variables from multiple scales when quantifying species responses to landscape modification.

Reproductive foragers: male spiders choose mates by selecting among competitive environments.

Mate choice frequently operates differently for males and females as a consequence of male competition for mates. Competitive interactions can alter the fitness payoffs of choice and the realization of preferences under natural conditions, yet the majority of male choice studies still use binary trials that ignore social factors. Here we test the importance of contest dynamics in male choice using a framework in which females are considered analogous to foraging patches that are subject to competition. We track the mate choices and interactions of 640 spiders (Nephila plumipes) before and after manipulation of competition within enclosures, modeling the expected fitness payoffs of each male's actual choices and comparing these with all alternative choices. Many males choose new mates once social conditions change and achieve higher fitness than predicted under random movement. Males do not simply move to larger females but choose favorable competitive environments that balance competition and female fecundity, thereby increasing their fitness payoffs. Further, we show for the first time that prior-residence effects, which are known to influence male contests, also have a strong influence in male reproductive contests and can shape male mate choice. These results highlight the importance of situating male choice studies in the relevant social context, as intrasexual interactions can have profound effects on the realization and payoffs of male mate-choice strategies.

Social Factors Driving Settlement and Relocation Decisions in a Solitary and Aggregative Spider

Both ecological and social factors play an important role in determining the structure of animal settlement patterns. While the ecological factors determining animal settlement are generally well known, the relative importance of social factors in mediating fine-scale settlement choices is poorly understood. As a result, we have little knowledge of why individuals choose to settle near specific neighbors. Here we used a web-building spider (Nephila plumipes) that settles both solitarily and next to neighbors within aggregations to examine the specific social factors that influence settlement decisions. Within experimental enclosures, we observed the settlement patterns of females pre- and postmale release. This allowed us to compare two models of aggregative settlement in lekking species-the hotshot and preferences models-to examine the relative importance of a female's phenotype and mate attraction to further dissect settlement and relocation decisions. We show that mate attraction increased with aggregation size and that larger females were generally preferred, supporting both the hotshot and preference models of aggregative settlement. We further demonstrate that smaller females that attracted fewer males within an aggregation were most likely to relocate. Our results demonstrate how social feedback can affect initially state-dependent settlement decisions, thereby highlighting the dynamic nature of settlement.

The active metabolic rate predicts a male spider's proximity to females and expected fitness

Conspicuous traits, such as weaponry and body size, are often correlated with fitness. By contrast, we understand less about how inconspicuous physiological traits affect fitness. Not only is linking physiology directly to fitness a challenge, but in addition, behavioural studies most often focus on resting or basal metabolic rates, resulting in a poor understanding of how active metabolic rates affect fitness. Here we use the golden orb-web spider (Nephila plumipes), a species for which proximity to a female on the web predicts a male's paternity share, to examine the role of resting and active metabolic rates in fitness. Using a semi-natural experimental set-up, we show that males closer to a female have higher active metabolic rates than males further from females. This higher metabolic activity is paralleled by increased citrate synthase activity, suggesting greater mitochondrial densities. Our results link both higher active metabolic rates and increased citrate synthase activity with fitness. Coupled with the behaviour and life history of N. plumipes, these results provide insight into the evolution of physiological systems.

Use of spider silk for nest building by the Regent HoneyeaterAnthochaera phrygiaand the Helmeted HoneyeaterLichenostomus melanops cassidix

The Regent Honeyeater Anthochaera phrygia and Helmeted Honeyeater Lichenostomus melanops cassidix have both suffered a dramatic decline in number and reduction in range as a result of extensive habitat clearance. As part of recovery plans aimed toward reintroduction to the wild, both species are part of an intensive captive breeding program being conducted at a number of institutions, including Taronga Zoo, Sydney. These honeyeaters incorporate arthropod silk into their nests, as has been reported for other small passerines. However, little is known about how birds choose and use the silk that they collect during nest building. We presented breeding pairs with cribellate silk from the Black House Spider Badumna sp. and ecribellate silk from the Golden Orb-weaver Nephila plumipes or ‘clean/fresh’ and ‘dirty/old’ Badumna sp. silk and observed which were taken by the birds. Lichenostomus melanops cassidix showed a preference for the cribellate silk of Badumna sp., always selecting this silk type first. Never...

Whole-organism performance and repeatability of locomotion on inclines in spiders

Burst speed, a common metric of locomotor performance, is typically measured on horizontal surfaces. Studies of locomotor performance in spiders have examined vertical and horizontal locomotion, but not the effects of intermediate inclines. We measured the effect of angle of incline, body size, relative leg length (variation not explained by body size) and relative body mass (variation not explained by body size or leg length, often interpreted as ‘condition’) on maximum running speed in two Australian spiders differing in habitat architecture and foraging strategy. Males of a ground-dwelling jumping spider, Jacksonoides queenslandica, and an orb-web spider, Nephila plumipes, were forced to run on raceways inclined at +0°, +30° and +60°. At the population level, maximum running speed decreased on inclined compared to level raceways for both species, but the effects of slope varied substantially between individuals. While speed was not influenced by body size or relative mass in either species, it was influenced by leg length in both. In J. queenslandica, relative leg length did not influence horizontal running speed but was positively related to speed on slopes, principally because relatively short-legged spiders were slowed whereas relatively long-legged spiders were not affected. In N. plumipes relative leg length was related negatively to running speed on horizontal surfaces but positively to running speed on slopes. Unlike J. queenslandica, N. plumipes was little affected by slopes of 30° but slowed markedly on slopes of 60°, irrespective of relative leg length. We suggest that performance advantages on inclines may promote relatively longer legs.

Functional relations between locomotor performance traits in spiders and implications for evolutionary hypotheses.

BackgroundLocomotor performance in ecologically relevant activities is often linked to individual fitness. Recent controversy over evolution of extreme sexual size dimorphism (SSD) in spiders centres on the relationship between size and locomotor capacity in males. Advantages for large males running over horizontal surfaces and small males climbing vertically have been proposed. Models have implicitly treated running and climbing as functionally distinct activities and failed to consider the possibility that they reflect common underlying capacities.FindingsWe examine the relationship between maximum climbing and running performance in males of three spider species. Maximum running and climbing speeds were positively related in two orb-web spiders with high SSD (Argiope keyserlingi and Nephila plumipes), indicating that for these species assays of running and climbing largely reveal the same underlying capacities. Running and climbing speeds were not related in a jumping spider with low SSD (Jacksonoides queenslandica). We found no evidence of a performance trade-off between these activities.ConclusionsIn the web-spiders A. keyserlingi and N. plumipes good runners were also good climbers. This indicates that climbing and running largely represent a single locomotor performance characteristic in these spiders, but this was not the case for the jumping spider J. queenslandica. There was no evidence of a trade-off between maximum running and climbing speeds in these spiders. We highlight the need to establish the relationship between apparently disparate locomotor activities when testing alternative hypotheses that yield predictions about different locomotor activities. Analysis of slopes suggests greater potential for an evolutionary response on performance in the horizontal compared to vertical context in these spiders.

The effects of morphology and substrate diameter on climbing and locomotor performance in male spiders

Summary 1. Spiders are the most sexually size dimorphic terrestrial animals and the evolution of this dimorphism is controversial. Patterns of sexual size dimorphism (SSD) in spiders have been related to individual performance and size. In 2002 Moya‐Laraño, Halaj & Wise proposed the ‘gravity hypothesis’ to explain patterns of sexual size dimorphism in spiders whereby species building webs high in the vegetation are predicted to show greater SSD than those that build lower down. They advocated an advantage in climbing speed in smaller males searching for females in high places. The gravity hypothesis predicts a negative relationship between male size and climbing speed. In 2007 Brandt & Andrade questioned this interpretation and proposed that the pattern of SSD in spiders is better explained by an advantage for larger males of low‐dwelling species to run faster along the ground. 2. We induced male spiders to run a standard distance up vertical poles of different diameters to examine the predicted relationship between size and climbing speed. We tested two species of extremely size‐dimorphic orb‐web spiders, Argiope keyserlingi and Nephila plumipes, that differ in the height at which females tend to build webs, and one species of jumping spider, Jacksonoides queenslandica, with low levels of size dimorphism. We also examined morphological determinants of horizontal motility by inducing males to run along a raceway. 3. Substrate diameter was consistently found to influence climbing performance. In N. plumipes, climbing speed was slowest on the widest diameter substrate. In A. keyserlingi, size‐adjusted leg length and substrate diameter interacted to determine climbing speed, while in J. queenslandica, there was an interaction between body size and substrate diameter on climbing speed. In the effect of substrate diameter, we have identified a potential bias in previous tests of the gravity hypothesis. 4. Our results do not support the prediction of the gravity hypothesis. There was no evidence of a negative relationship between body size and climbing speed in the two orb‐web species with high levels of SSD. Our results are also not consistent with a recent modification of the gravity hypothesis that suggests a curvilinear relationship between climbing speed and size. 5. Body size was positively associated with maximum running speed only in the cursorial hunter J. queenslandica. For this spider, results are more consistent with Brandt & Andrade’s explanation for variation in SSD in spiders, that larger males are selected for superior running ability in low‐dwelling species, rather than selection for smaller size for climbing to females in high‐dwelling species.

Evidence for developmental plasticity in response to demographic variation in nature

Adaptive developmental plasticity has been demonstrated across a number of taxa in response to variables such as photoperiod, resource abundance, and predator presence. Demographics also vary temporally and spatially within populations, but few studies have examined the possibility that developmental plasticity in response to changes in these variables can alter phenotypic distributions. Plastic responses to variable population density and sex ratio may play an important role in explaining phenotypic variation in nature. In this study, we examine two species of spiders (Nephila plumipes and Argiope keyserlingi) to examine whether there is evidence that males alter their development in response to demographics in natural populations. We studied spiders in which developing males can use pheromones as a cue of the density of conspecific males and females. We used published information about the mating systems and life history of each species to make predictions about expected patterns of plasticity in development time and correlated changes in adult body size in response to demographic variation. Within each species, male size and mass were positively correlated with the density of males but negatively correlated with the density of females, and as predicted, this was true only when calculated at spatial scales relevant to selection in each species. In contrast, seasonal variation in photoperiod could not explain measured variance in male size. Our results support the idea that developmental plasticity in response to demographics has a significant effect on phenotypic distributions in natural populations. Our results suggest that a proportion of variation in male phenotypes could be explained as a plasticity-mediated evolutionary response to variation in population demographics rather than as a physiological response to resource abundance and/or photoperiod.

SPATIAL AND TEMPORAL DEMOGRAPHIC VARIATION DRIVES WITHIN-SEASON FLUCTUATIONS IN SEXUAL SELECTION

Our understanding of selection in nature stems mainly from whole-season and cross-sectional estimates of selection gradients. These estimates suggest that selection is relatively constant within, but fluctuates between seasons. However, the strength of selection depends on demographics, and because demographics can vary within seasons, there is a gap in our understanding regarding the extent to which seasonal fluctuations in demographics may cause variation in selection. Here we use two populations of the golden orb-web spider (Nephila plumipes) that differ in density to examine how demographics change within a season and whether there are correlated shifts in selection. We demonstrate that there is within-season variation in sex ratio and density at multiple spatial and temporal scales. This variation led to changes in the competitive challenges that males encountered at different times of the season and was correlated with significant variation in selection gradients on male size and weight between sampling periods. We highlight the importance of understanding the biology of the organism under study to correctly determine the relevant scale in which to examine selection. We also argue that studies may underestimate the true variation in selection by averaging values, leading to misinterpretation of the effect of selection on phenotypic evolution.

Male copulation frequency, sperm competition and genital damage in the golden orb-web spider (Nephila plumipes)

Copulation in many sexually cannibalistic spiders is associated with a loss of function of the male reproductive organs and, as a consequence, males that survive sexual cannibalism may nevertheless be unable to subsequently copulate successfully. Sexual cannibalism is common in the Australian golden orb-web spider (Nephila plumipes), in which the tip of the conductor typically breaks during copulation. Thus, male mating frequency may be physiologically limited to two females, irrespective of the male’s ability to avoid cannibalism or the opportunity to locate and court additional, receptive females. Laboratory experiments revealed that the likelihood of the conductor breaking depends upon the copulatory history of the female insemination duct: males were more likely to break their conductor if they inseminated a ‘virgin’ rather than ‘mated’ insemination duct. However, the choice of insemination duct did not influence the duration of copulation or quantity of sperm transferred. In field populations, the proportion of males with both conductors broken increased during the course of the mating season, but while males with broken conductors did not copulate successfully with virgin females, they were nevertheless observed on the webs of immature females. We suggest that male N. plumipes with broken conductors on the webs of females are most likely mate guarding, as this appears to be the most effective mechanism of securing paternity.

The systematics and biology of the spider genus Nephila (Araneae:Nephilidae) in the Australasian region

Five species of the nephilid genus Nephila Leach are found in the Australasian region, which for the purposes of this study was defined as Australia and its dependencies (including Lord Howe I., Norfolk I., Christmas I., Cocos (Keeling) Is), New Guinea (including Papua New Guinea and the Indonesian province of West Papua), Solomon Is, Vanuatu, New Caledonia, Fiji, Tonga, Niue, New Zealand and other parts of the south-west Pacific region. All species are redescribed and illustrated. Nephila pilipes (Fabricius) occurs in the closed forests of eastern and northern Australia, New Guinea, Solomon Is and Vanuatu (through to South-East Asia); N. plumipes (Latreille) is found in Australia (including Lord Howe I. and Norfolk I.), New Guinea, Vanuatu, Solomon Is and New Caledonia; N. tetragnathoides (Walckenaer) inhabits Fiji, Tonga and Niue; N. antipodiana (Walckenaer) occurs in northern Australia (as well as Christmas I.), New Guinea and Solomon Is (through to South-East Asia); and N. edulis (Labillardière) is found in Australia (including Cocos (Keeling) Is), New Guinea, New Zealand and New Caledonia. Epeira (Nephila) walckenaeri Doleschall, E. (N.) hasseltii Doleschall, N. maculata var. annulipes Thorell, N. maculata jalorensis Simon, N. maculata var. novae-guineae Strand, N. pictithorax Kulczyński, N. maculata var. flavornata Merian, N. pictithorax Kulczyński, N. maculata var. flavornata Merian, N. maculata piscatorum de Vis, and N. (N.) maculata var. lauterbachi Dahl are proposed as new synonyms of N. pilipes. Nephila imperialis var. novaemecklenburgiae Strand, N. ambigua Kulczyński, N. sarasinorum Merian and N. celebesiana Strand are proposed as new synonyms of N. antipodiana. Meta aerea Hogg, N. meridionalis Hogg, N. adelaidensis Hogg and N. meridionalis hermitis Hogg are proposed as new synonyms of N. edulis. Nephila picta Rainbow is removed from the synonymy of N. plumipes and treated as a synonym of N. edulis, and N. nigritarsis insulicola Pocock is removed from the synonymy of N. plumipes and treated as a synonym of N. antipodiana. Allozyme data demonstrate that N. pilipes is distinct at the 80% FD level from N. edulis, N. plumipes and N. tetragnathoides. Nephila plumipes and N. tetragnathoides, deemed to represent sister-taxa owing to the shared presence of a triangular protrusion of the male pedipalpal conductor, were found to differ at 15% FD in the genetic study. No genetic differentiation was found between 10 populations of N. edulis sampled across mainland Australia. Species of the genus Nephila have been extensively used in ecological and behavioural studies, and the biology of Nephila species in the Australasian region is extensively reviewed and compared with studies on Nephila species from other regions of the world.

Risky mate search and mate preference in the golden orb-web spider (Nephila plumipes)

Mate searching is a risky behavior that decreases survival by increasing predation risk and the risk of energy depletion. However, few studies have quantified actual mortality during mate search, making it difficult to predict mate searching and mating strategies. Using a mark and recapture study, we examined mate-searching success in a highly sexually dimorphic species, the golden orb-web spider (Nephila plumipes). We show that despite the high-density aggregations of this species, male survival during mate searching is extremely low (36%) and is phenotype independent. Surprisingly, males that survived mate search were in better condition after recapture than prior to release, most likely due to kleptoparasitism on females' webs. In a complementary release experiment in a field enclosure, we show that males are choosy and adjust their choice of female depending on their own condition and weight. Thus, the high mortality rate of searching males in the field may be a cost of choosiness because released males traveled further than necessary to settle on females. Although males were choosy about female phenotypes, they did not avoid webs with rival males already present. This suggests that the cost of continued searching outweighs the cost of competition but not the cost of mating with certain females. Further examinations of mate-searching risk in other species in reference to their mating system and environmental conditions are necessary to determine the occurrence and effects of high mortality rates during searching.

Frequency, composition and variation in external food stores constructed by orb-web spiders: Nephila edulis and Nephila plumipes (Araneae : Araneoidea)

Few examples of food hoarding have been documented in spiders, yet two Australian species of orb-web spiders, Nephila edulis and N. plumipes, typically incorporate previously captured prey into the web. The effect of prey density and prey-encounter rate on the storage behaviour of adult female N. edulis was tested in the laboratory. Prey density had a significant effect on the propensity to construct external caches: when more food was available, food caches were larger than when the supply of prey was limited. Caching behaviour also differed with the rate of prey encounter, even though the total amount of food supplied was the same. When prey were encountered at constant rates, spiders allocated more food to external storage compared with random encounter rates. Finally, we tested the quality of different prey types for external or internal storage. N. edulis were fed with blow-flies or crickets, and these prey were stored in the web, discarded or totally consumed. Crickets were typically consumed or stored, while flies were more frequently discarded. Field observations of the storage behaviour in N. edulis and N. plumipes found surprising differences in the composition of the cache. While N. plumipes incorporated only animal material, N. edulis also utilised plant material, suggesting that the storage band in N. edulis has other, non-food-storing functions. Field experiments indicated that the presence or absence of external stores in the web of N. plumipes had no influence on mortality, weight gain, or the presence of Argyrodes kleptoparasites.