Abstract
Polymerase chain reaction (PCR) gut analysis was conducted on specimens of the introduced spider Tenuiphantes tenuis collected from dairy pasture in Canterbury, New Zealand. PCR primers were specifically designed to amplify a fragment of the mitochondrial gene cytochrome c oxidase subunit 1 (COI) from Listronotus bonariensis and revealed that this major pasture pest species is consumed in the field by T. tenuis. The field predation rate of L. bonariensis by T. tenuis was estimated from our PCR results together with published data on the degradation of DNA and the density of T. tenuis in Canterbury pastures. We found that T. tenuis is a potentially significant predator of L. bonariensis in New Zealand pastures.
Introduction
Tenuiphantes tenuis (Blackwall, 1852) is a European spider in the family Linyphiidae that has been introduced to New Zealand, the Azores, Madeira, the Canary Islands, Canada (British Columbia, Newfoundland), USA (Hawaii, Washington. Oregon, Massachusetts), Chile and Argentina (van Helsdingen et al. Citation1977; Millidge Citation1988, Citation1991; Beatty et al. Citation2000; Buckle et al. Citation2001; Paquin et al. Citation2010b). It was first noted in New Zealand by Forster & Forster (Citation1973), by which time it was already widespread. Tenuiphantes tenuis was probably introduced to New Zealand in colonial times when several other European linyphiid species were also thought to have been accidentally introduced (Millidge Citation1988). Tenuiphantes tenuis is a small (2–2.5 mm body length) spider that is widespread and common throughout New Zealand, particularly in the South Island (Millidge Citation1988). It is found in high numbers in pasture and arable crops (Martin Citation1983; Millidge Citation1988; Sivasubramaniam et al. Citation1997; Topping & Lövei Citation1997; McLachlan & Wratten Citation2003; Clark et al. Citation2004; Vink et al. Citation2004) and also occurs in disturbed native forest and pine plantations (Millidge Citation1988). In Canterbury, it is the numerically dominant spider species in agricultural habitats (Sivasubramaniam et al. Citation1997; McLachlan & Wratten Citation2003; Vink et al. Citation2004) where it occurs in higher numbers than other arthropod predators (Sivasubramaniam et al. Citation1997). Up to 79 T. tenuis m−2 have been found in pasture in Canterbury (Vink et al. Citation2004). Despite the abundance of T. tenuis in New Zealand agroecosystems, nothing is known about what it feeds on there. In the UK and Belgium, T. tenuis has been shown to feed on aphid pests and Collembola (Sunderland et al. Citation1986a, Citation1987; Alderweireldt Citation1994).
Tenuiphantes tenuis uses a horizontal sheet web to capture prey, but rarely succeeds in capturing and killing prey that is over 5 mm long (Harwood et al. Citation2001). There are a number of pest species in New Zealand pasture that are less than 5 mm long and most notable is Listronotus bonariensis (Kuschel, 1955), the Argentine stem weevil. This species was first discovered in New Zealand in 1927 (Marshall Citation1938; Kuschel Citation1972) and is one of New Zealand's most damaging pasture pests (Prestidge et al. Citation1991). Listronotus bonariensis feeds on pasture grasses, especially ryegrass, Lolium spp., as well as other graminaceous crops. Chemical control of L. bonariensis in pasture is difficult as early larval instars feed inside grass stems and pupate below the soil (Chapman Citation1984). Later instars can be found both in the tillers and thatch with up to 27% of fourth-instar larvae found in the thatch (Goldson et al. Citation2001). In 1991, the parasitoid Microctonus hyperodae Loan, 1974 was introduced as a biological control for L. bonariensis and appeared to be successful in suppressing L. bonariensis (Goldson et al. Citation1998; McNeill et al. Citation2002; Barker & Addison Citation2006). However, recent research has indicated that L. bonariensis can still cause major damage to susceptible ryegrass pastures (Popay et al. Citation2011); therefore other biological control methods may be necessary.
Generalist predators can reduce pest numbers significantly (Symondson et al. Citation2002) and because of the abundance of T. tenuis in New Zealand pasture, this species has the potential to help control L. bonariensis and other pasture pests. PCR (polymerase chain reaction) analyses of gut content has become an important tool in determining the diet of arthropod predators (Symondson Citation2002; Harwood & Greenstone Citation2009) and has been used to study predation by T. tenuis (Sheppard et al. Citation2005) and other linyphiid spiders (e.g. Chapman et al. Citation2013). This method is used here to determine whether T. tenuis feeds on L. bonariensis in a pasture in Canterbury, New Zealand, and is the first time empirical data has been used to investigate the prey of a pasture inhabiting predacious arthropod in New Zealand.
Methods
We used the series of methods outlined in Vink et al. (Citation2011) in this study.
DNA extraction and PCR amplification
DNA was extracted using a Quick-gDNA™ MiniPrep kit (Zymo Research) from two to three legs of the specimens collected for sequence variation determination. A fragment of the mitochondrial gene cytochrome c oxidase subunit 1 (COI) was PCR amplified and sequenced for T. tenuis and L. bonariensis. For T. tenuis, the primer pair LCO-1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) (Folmer et al. Citation1994) plus C1-N-2776-spider (5′-GGATAATCAGAATANCGNCGAGG-3′) (Vink et al. Citation2005) was used to amplify a 1261-base pair (bp) fragment. However, this primer pair did not always work, in which case the primer pair C1-J-1718-spider (5′-GGNGGATTTGGAAATTGRTTRGTTCC-3′) (Vink et al. Citation2005) plus C1-N-2568 (Hedin & Maddison Citation2001) was used to amplify an 850-bp fragment. For L. bonariensis, the primer pair LCO1490 plus C1-N-2650-weevil (5′-CCNGTRAATARNGGGAATCATTG-3′) (Vink & Phillips Citation2007) was used to amplify an 1135-bp fragment. PCR amplification was performed using i-StarTaq™ DNA Polymerase (iNtRON Biotechnology) in a Mastercycler® (Eppendorf) thermocycler with a cycling profile of 35 cycles of 94 °C denaturation (30 s), 48 °C annealing (30 s), 72 °C extension (1 min) with an initial denaturation of 3 min and a final extension of 5 min. Excess primers and salts were removed from the resulting double-stranded DNA using a DNA Clean & Concentrator™ Kit (Zymo Research). Purified PCR fragments of DNA were sequenced in both directions at either Macrogen (Seoul) or the Massey Genome Service (Massey University). Sequence data were deposited in GenBank (www.ncbi.nlm.nih.gov/Genbank/). Sequences were edited and compared with each other using Sequencher 4.6 (Gene Codes Corporation).
Sequence variation
In order to design primers that were specific to all possible COI haplotypes of L. bonariensis found in New Zealand, sequence variation was determined from 13 specimens that had been collected from eight locations throughout New Zealand (). To ensure specific primers were designed not to amplify T. tenuis DNA, sequence variation in T. tenuis was also investigated in 11 specimens that had been collected from six locations throughout New Zealand (). Specimens of T. tenuis were morphologically identified using Millidge (Citation1988) and Paquin et al. (Citation2010a). Listronotus bonariensis is a well-known pest in New Zealand, and therefore identification was trivial; however, a key in Kuschel (Citation1972) can be used to identify adults, and larvae can be identified using May (Citation1993).
Table 1 Collecting information for specimens; two-letter area codes follow Crosby et al. (Citation1998).
Specific primer design and testing
Using L. bonariensis COI sequences as a reference, primers were designed to specifically amplify a short (130-bp) fragment of L. bonariensis COI DNA. Prey DNA detection is more successful when short fragments are targeted (King et al. Citation2008) and shorter fragments persist for longer in the guts of T. tenuis (Sheppard et al. Citation2005). Primers were designed using an online version of Primer3 (Rozen & Skaletsky Citation2000). These primers (C1-J-1850-ASW (5′-GCAGGGACTGGATGAACAGT-3′) and C1-N-1981-ASW (5′-GGCGTCATACCTTCTGGGTA-3′)) were then compared with other COI sequences of spiders (e.g. Vink Citation2002; Vink et al. Citation2009) and pasture pests (e.g. Vink & Phillips Citation2007) to ensure that they would not anneal non-target COI DNA.
The primers specific to L. bonariensis (C1-J-1850-ASW plus C1-N-1981-ASW) were tested on DNA extracted from two to three legs of arthropods commonly found in pasture that were less than 5 mm long as either adults or immature stages. The following species were tested: the spiders Erigone prominens Bosenberg & Strand, 1906, Erigone wiltoni Locket, 1973, Anoteropsis hilaris (L. Koch, 1877) and Cryptachaea blattea (Urquhart 1886); the weevils Sitona lepidus Gyllenhal 1834, Sitona discoideus Gyllenhal 1834, Exapion ulicis (Forster 1771); the Collembola Sminthurus viridis (Linnaeus 1758), Hypogastrura sp., Entomobrya sp.; and the aphid Acyrthosiphon pisum Harris, 1776. Species were identified using Kuschel (Citation1972), Millidge (Citation1988), Boulding (Citation1998), Vink (Citation2002), Phillips & Barratt (Citation2004), Hopkin (Citation2007) and Vink et al. (Citation2009). DNA extracted from L. bonariensis and T. tenuis was also tested as a positive and negative control, respectively. PCR conditions were as described above except for the annealing temperature, which was initially set at 51 °C, but was raised to 55 °C to eliminate weak false positives in E. ulicis DNA.
The concentration of L. bonariensis DNA extractions was measured on a Thermo Scientific NanoDrop 2000 spectrophotometer; a sample of eluted DNA was then diluted 10-fold sequentially to ascertain the minimum quantity of DNA detectible using the primers specific to L. bonariensis.
PCR gut analysis of field collected spiders
DNA was extracted from 67 entire specimens of T. tenuis [54 females (adult and subadult) and 13 males] that had been collected in a dairy pasture at Swannanoa (43°25.7'S, 172°27.1'E) on the morning of 17 February 2010, a time of year when L. bonariensis adult and larval densities are typically high (McNeill et al. Citation2001). Specimens were collected using a suction sampler (Echo ES-2400, 24 cm3, Kioritz Corporation, Tokyo), with a detachable net fitted into the inlet. Tenuiphates tenuis specimens were then removed from the net by hand, immediately stored in 95% ethanol in separate vials and kept at −20 °C, using a Nalgene® Labtop Cooler to minimise DNA degradation (Vink et al. Citation2005). DNA was extracted from entire T. tenuis specimens using the methods above. PCR conditions were as described above except for the annealing temperature; which was 55 °C. DNA extracted from a specimen of L. bonariensis was run as a positive control that also confirmed the correct fragment size when results were visualised via gel electrophoresis. To eliminate the possibility that DNA from untested prey species may cause false positives, we sequenced DNA from each appropriate-sized band (170 bp, including primer annealing sites), which was isolated using a Zymoclean™ Gel DNA Recovery kit to remove the excess primers and salts. Purified PCR fragments were sequenced in both directions at Macrogen (Seoul). Sequences were assembled and edited in Sequencher 4.6 (Gene Codes Corporation) and compared with L. bonariensis sequences.
Adult and subadult female specimens of T. tenuis were preferentially collected as male spiders of many species do not catch prey after achieving their final moult (Foelix Citation2011); however, we did collect 13 males to see if there was any difference in the ratio of female and male T. tenuis that tested positive for the presence of L. bonariensis DNA. A chi-squared test was conducted with an a priori expectation that there would be no difference in the ratio.
Predation rate calculation
Sheppard et al. (Citation2005) fitted regression lines to the temporal decline in detectability of 155- and 242-bp amplicons of aphid DNA in T. tenius gut samples. However, Sheppard et al. (Citation2005) incorrectly reported the amplicon sizes as 110 and 245 bp, respectively; they did not include the primer annealing sites for the shorter amplicon and the longer amplicon was 3 bp shorter than reported by Harper et al. (Citation2005). The corrected amplicon sizes we report are based on published sequences of Sitobion avenae (Fabricius, 1775) (Agustí et al. Citation2003; Foottit et al. Citation2008; King et al. Citation2011).
It is possible that S. avenae DNA is digested at a different rate from L. bonariensis DNA and ideally feeding trials using T. tenius and L. bonariensis should be used to estimate the true rate of DNA degradation. However, it seems unlikely that the rate of digestion of our fragment of L. bonariensis DNA would differ markedly from the fragments reported in Sheppard et al. (Citation2005). Our 170-bp amplicon is from a similar part of COI as the amplicons in Sheppard et al. (Citation2005) and they have a 66-bp overlap. As the size of the amplicon in our study was between the sizes in Sheppard et al. (Citation2005), we interpolated between the corrected regression lines in Sheppard et al. (Citation2005) for our 170-bp amplicon, suggesting p=1−0.21x, where p is the proportion (0 to 1) of spiders testing positive and x is the time since feeding in days. Given the observed proportion of spiders testing positive, we estimated the mean time t between L. bonariensis feeding events in our field samples by integration of this relationship:
Results
Three COI haplotypes were found in T. tenuis collected from six sites throughout New Zealand. Genetic divergences between the three haplotypes ranged from 0.3–1.7%. Only one haplotype of COI was found in all of the L. bonariensis specimens collected from eight sites throughout New Zealand.
PCR amplification was still successful using the specific primers C1-J-1850-ASW plus C1-N-1981-ASW when the template L. bonariensis was 1.5×10−4 ng/µl.
A 170-bp (including primer annealing sites) fragment of COI was amplified from 12 of the 67 specimens (17.9%) of T. tenuis, which were all females (adult or subadult). All fragments were confirmed as L. bonariensis by sequencing. From the model above, this suggests the mean time between feeding events on L. bonariensis is t=13.3 days.
In ryegrass (Lolium perenne L.) pasture, T. tenuis has been recorded in densities as high as 51.8 m−2 (Vink et al. Citation2004), which suggests predation rates of up to 3.9 L. bonariensis m−2 per day.
None of the 13 male T. tenuis tested positive for the presence of L. bonariensis DNA; however, a chi-squared test did not reveal a significant difference in the ratio of female and male positive results (χ2=2.891, v=1, P=0.0891).
Discussion
It is clear from our results that T. tenuis does feed on L. bonariensis in pasture in Canterbury. Our estimates of predation rate are very preliminary, as they are based on DNA digestion rates reported for another prey species (Sheppard et al. Citation2005) and the densities we used were from a previously study in Canterbury pastures (Vink et al. Citation2004). Ideally, these rates and densities should be measured concurrently as part of a larger study; however, taking into account the assumptions we made, dense T. tenuis populations could potentially remove 3.9 L. bonariensis m−2 per day. Typical L. bonariensis densities in Canterbury paddocks range from 100 to 400 adults m−2 at their peak in February (Goldson et al. Citation1998), which is also when T. tenuis numbers peak (Vink et al. Citation2004) and therefore it is feasible that T. tenuis could have a measurable effect on L. bonariensis populations. The magnitude of this population suppression is currently unknown, but simple models suggest that it will depend on the ability of the prey to compensate for offtake through its intrinsic rate of increase, modified by other density-dependent factors (Kean et al. Citation2003; Barratt et al. Citation2010).
As a generalist predator, T. tenuis may switch to alternative prey when L. bonariensis densities become low and so be unlikely to reduce L. bonariensis populations to the same extent as a specialist parasitoid, such as Microctonus hyperodae (Goldson et al. Citation1998; McNeill et al. Citation2002; Barker & Addison Citation2006). Further work would be required to assess the functional response of the prey consumption rate versus prey population size. Tenuiphantes tenuis can be found in high numbers from August to March (Vink et al. Citation2004), which encompasses all the times of the year when adult L. bonariensis are active in pasture (Goldson et al. Citation2011), and could help suppress L. bonariensis from spring to autumn. The ability of natural enemies to aggregate at local concentrations of prey may be an important factor determining the magnitude of prey suppression (Kean et al. Citation2003). Unlike many web building spiders, T. tenuis is very mobile and will relocate to where prey abundance is high (Harwood et al. Citation2003). Samu et al. (Citation1996) found that the mean duration of an individual T. tenuis in a web site is less than 2 days. Tenuiphantes tenuis is also thought to be preadapted to agricultural environments, as it disperses by ballooning on threads of silk to avoid adverse conditions (Topping & Sunderland Citation1998).
Tenuiphantes tenuis would be able to survive periods when L. bonariensis numbers were low as they can switch to alternative prey. In Europe, T. tenuis feeds on Collembola (Sunderland et al. Citation1986a; Alderweireldt Citation1994; Agustí et al. Citation2003) and the common collembolan species found in New Zealand pasture are introduced European species (Greenslade et al. Citationin press). Tenuiphantes tenuis is also a significant predator of aphids (Sunderland et al. Citation1987; Alderweireldt Citation1994), some species of which (e.g. Acyrthosiphon pisum) can also be pests in New Zealand pasture.
Several factors could affect our estimates of L. bonariensis predation. Suction samples have been shown to cross-contaminate predators with prey DNA (Greenstone et al. Citation2011), which could lead to false positives; however, L. bonariensis, is robust and is unlikely to rupture during suction sampling. We also minimised the time T. tenuis spend in the suction sampler to reduce the possibility of predation occurring during sampling.
Male spiders of many species do not catch prey after achieving their final moult and leave their web in search of females to mate with (Foelix Citation2011). It is unknown whether this is the case for T. tenuis. We did not find any L. bonariensis DNA in the 13 males we sampled; however, there was no significant difference in the ratio of females and males that tested positive for the presence of L. bonariensis DNA. Our sampling was biased towards collecting females and the sex ratio of T. tenius in the field is reported to be equal (Topping & Sunderland Citation1998), so it may be possible that only females and subadults of both sexes prey on L. bonariensis, which would reduce the rate of predation. When Vink et al. (Citation2004) estimated T. tenius densities, they did not include immature linyphiid specimens, which could have increased T. tenius density estimates by up to 30%; however, smaller immature T. tenius may not be capable of capturing and feeding on L. bonariensis.
We have assumed that each positive recording of L. bonariensis DNA in a T. tenius represents just one death of the prey species, but it is possible that a spider may feed on multiple specimens of L. bonariensis within a short time. It is also possible that more L. bonariensis are killed by immobilisation in T. tenius webs where the web-owner may be too satiated to feed or the web has been abandoned. Tenuiphantes tenuis has been noted to trap large numbers of insects in its horizontal sheet web (Sunderland et al. Citation1986b; Alderweireldt Citation1994) and Sunderland (Citation1999) suggested that spider webs may contribute to pest control even if not all insects trapped in webs are consumed.
Given the predation rates we have estimated, increasing T. tenuis numbers in pasture may help control L. bonariensis. Reviews of research in Europe have shown that spider density in agroecosystems can be increased by habitat diversification (Samu et al. Citation1999; Sunderland & Samu Citation2000). Research in New Zealand found that cultivation and grazing had a detrimental effect on populations of T. tenuis and other spiders (Clark et al. Citation2004). A study in England (Bell et al. Citation2002) indicates that taller and more structurally complex grassland vegetation increased T. tenuis population size and field margins provided an excellent source habitat for T. tenuis. Further studies comparing densities of T. tenuis in pastures of differing plant diversity coupled with density measures of L. bonariensis and predation rates based on PCR gut analysis could reveal whether T. tenuis can help suppress L. bonariensis populations. Ultimately, we aim to use PCR gut analysis to establish which New Zealand predacious arthropods in pasture are most beneficial and how to augment their suppression of pests using conservation biological control (Kean et al. Citation2003).
Acknowledgements
Thanks to Mark McNeill (AgResearch) for assistance in collecting T. tenuis specimens. Mark McNeill, Sean Marshall and two anonymous reviewers provided valuable comments on an earlier draft of the manuscript. This research was supported by from New Zealand's Ministry of Science and Innovation through contract LINX0304, Ecosystems Bioprotection.
Related Research Data
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