Bulletin of Insectology 63 (2): 203-208, 2010 ISSN 1721-8861 A survey of nectar feeding by spiders in three different habitats Xiaoqiong CHEN, Yuanchun CHEN, Lingbing WU, Yu PENG, Jian CHEN, Fengxiang LIU College of Life Sciences, Hubei University, Wuhan, People’s Republic of China Abstract Using cold-anthrone tests, spiders collected in the field were sampled for the presence of plant sugar (fructose). The spiders came from three different habitats: a cotton field, a mixed rape and fava bean field, and a park with flowering woody and herbaceous plants. The percentages of fructose-positive spiders were compared among the different habitats, as well as among the different sexes and ages of Ebrechtella tricuspidata (F.) (Araneae Thomisidae). Out of 745 field-collected spiders, 18.7% were positive for fructose, indicating that these spiders feed on plant nectar. Of the 12 families of spiders represented, individuals from 9 families were positive for fructose: Oxyopidae, Thomisidae, Pisauridae, Salticidae, Lycosidae, Tetragnathidae, Araneidae, Nephilidae, and Agelenidae. All members of the other three families (Linyphiidae, Clubionidae and Theridiidae) were negative for fructose. There were no differences for nectar feeding in spiders among the three habitats: 19.3% of individuals from the cotton field, 16.6% from the mixed rape and fava bean field, and 20.0% in park with flowering woody and herbaceous plants tested positive for fructose. For E. tricuspidata, significantly more females were positive than males (87.5% versus 42.9%); and immatures tested positive at a lower rate than adults (26.5% and 66.7%, respectively). Key words: spider, nectar, fructose, anthrone-sulphuric acid. Introduction proportions (Nicolson and Thornburg, 2007). A test for fructose can ensure that the sugar is from plants, distin- Spiders are generally envisaged as being obligate carni- guishing it from glucose. Although the latter is a com- vores (Nentwig, 1987; Foelix, 1996), meaning that they ponent of nectar, it also occurs in the blood and lym- acquire nutrition by feeding on other invertebrates. They phatic elements of some spiders (Barron et al., 1999). are especially known for feeding on other active arthro- Reacting with concentrated sulphuric acid, fructose is pods, but some spiders eat insect eggs or the eggs of o- dehydrated to generate furfural or hydroxymethylfur- ther spiders (Buschman et al., 1977; Willey and Adler, fural, which can react with anthrone to generate blue- 1989; Nyffeler et al., 1990; Jackson and Willey, 1994; green or blue furfural derivatives. The depth of colour is Cross et al., 2009). There are also instances of spiders proportional to the fructose content with a certain scope, feeding on dead arthropods (Sandidge, 2003). However, so the anthrone generated can be used to assess the spiders can also expand their food source by feeding on amount of fructose present in spiders. plants. Meehan et al. (2009) discovered herbivory in In the present study, we used the cold anthrone- Bagheera kiplingi Peckham et Peckham, a Neotropical sulphuric acid method to determine the concentration of jumping spider (Salticidae). The spider feeds primarily fructose in individual spiders, including web-building and deliberately on the tips of an ant-acacia leaflet. Sev- spiders and wandering spiders, collected from three dif- eral species of cursorial spiders imbibe nectar as an oc- ferent habitats: cotton field, a mixed rape and fava bean casional supplement to animal prey (Jackson et al., field, and a park with flowering woody and herbaceous 2001), and some juvenile orb-web spiders ingest pollen plants. Ebrechtella tricuspidata (F.) (Araneae Thomisi- when recycling their webs (Smith and Mommsen, dae), a dominant spider in the fields, was used as an ex- 1984). In addition, nectaries of plants including floral ample to compare the difference in nectar feeding and extrafloral nectaries (EFNs) are often visited by among spiders of different ages and sexes. wandering spiders, including members of Salticidae (Edmunds, 1978), Thomisidae (Vogelei and Greissl, 1989; Beck and Connor, 1992; Pollard et al., 1995), and Materials and methods the fast-moving nocturnal spiders Clubionidae (Ruhren and Handel, 1999), Anyphaenidae, and Corinnidae (Ta- Solution preparation ylor and Foster, 1996; Patt and Pfannenstiel, 2008). The anthrone reagent was prepared by pouring 380 ml When the spiders are near the nectaries, they can prey concentrated sulphuric acid into 150 ml distilled water on nectarivorous insects or can also feed directly on (diluted sulphuric acid), and then mixing 150 mg an- nectar. In these two ways, the spiders can extend their throne with 100 ml the diluted sulphuric acid. We cre- diets to an additional trophic level. ated a series of standard sucrose solutions correspond- We used the cold anthrone-sulphuric acid method ing to 1, 2, 4, 8, 16, 32, 64, 128, and 256 µg/µl (0.1- (Van Handel, 1972; Li et al., 2003; Taylor and Pfannen- 25.6% solutions) by dissolving 25.6 g of reagent-grade stiel, 2008), to determine the concentration of fructose sucrose in 50 ml of distilled water, adding enough water in spiders to judge whether they were feeding on nectar. to make 100 ml of solution, and making eight two-fold Fructose is a component of sucrose. Fructose, sucrose, serial dilutions (Haramis and Foster, 1983). These solu- and glucose can be found in plant nectar, in different tions were stored at -45 °C. The colorimetric standards for measuring fructose in the spider samples were cre- colorimetric standards and to determine the concentra- ated by pipetting 1 µl of nine standard sucrose solutions tions of fructose in the spiders. Evaluation of the centri- into a test tube and adding two drops of the 1:1 chloro- fuged and decanted sample by spectrophotometer was form-methanol solution and 0.5 ml of anthrone reagent. not possible because of the presence of spider remains The mixture solutions were held in a 38 °C water bath in the sample. Visual inspection without centrifuging for 1 h. The colour of the solutions changes from green allowed us to detect the content of fructose as low as 1 to green-blue in the presence of fructose, depending on µg. However, we included only readings of more than 2 the amount of fructose. We are able to identify fructose µg, to ensure that no false positives were recorded. This content as low as 1 µg. precaution also contributes to obtain a conservative es- timate of fructose-positive spiders. In this experiment, The collection and preparation of spiders the sample without fructose is a transparent yellow. If We collected spider samples early in the evening green was produced, it indicated only the presence or (18:00-21:00), between November 2008 and September existence of a combination of fructose and glucose 2009, from three different habitats in Hubei Province, formed by sucrose. Positive spiders either contained the China: a cotton field (CF) at Huazhong Agricultural same fructose content as the standard solutions, or as a University; a mixed rape and fava bean field (MRFBF) half of the standard solutions (with glucose). at the Oil Crops Research Institute, Chinese Academy of Agricultural Sciences; and a park with flowering wo- Data analysis ody and herbaceous plants (PFWHP) at Hubei Univer- The percentages of fructose-positive spiders were sity. A sweep net and glass tube (2 × 10 cm) were used compared among the three habitats by using signifi- to capture the spiders. The collected spiders were killed cance testing for cross-tabulation tables (2 × 3). Differ- immediately by immersion in liquid nitrogen, and were ences in positive percentages between sexes and ages of stored in a freezer at -86 °C. Because some digestion E. tricuspidata were compared by using significance most likely occurred between collection and complete testing for cross-tabulation tables (2 × 2). Chi-squares arrest of the enzymatic activity, our results should pro- (χ2) were calculated in SPSS.PASW.Statistics.v18. vide a conservative measure of nectar feeding in the field. Within a day or two, samples were heat-treated at 80-90 °C for 15 min, using a water bath to drive off all Results moisture, and were stored for later testing. Percentage of nectar feeding by individual spiders The detection of fructose in spiders From November 2008 through September 2009, 745 To prepare spiders for testing with anthrone, each spi- individual spiders were collected and were tested for der was placed in a 5-ml test tube and moistened with ingested fructose, including all ages and both sexes. A two drops of chloroform-methanol solution to remove total of 139 spiders or 18.7% tested positive for fructose cuticular wax. After 20 min, the spiders were gently (table 1). The fructose contents from 1 µg to 64 µg. To crushed with a glass stirring rod. Anthrone reagent (0.5 avoid false positives, our analysis included only with 2 ml) was added, and the tubes were agitated on a vortex µg fructose and above. 83.5% of the samples contained mixer, held in a 26 °C water bath for 1 h. The upper, amounts of fructose from 2 µg to 4 µg. The highest clear liquid was used to compare the colour with the readings appeared in two samples of two species: one Table 1. Percentage of fructose-positive individuals from 12 families of spiders in China (from November 2008 through September 2009). Percent (no. fructose-positive / no. spiders tested) Families Species of spider Adults + subadults Immatures Total Oxyopidae Oxyopes sertatus 48.7% (18/37) 30.0% (3/10) 44.7% (21/47) Thomisidae Ebrechtella tricuspidata 66.7% (46/69) 26.5% (39/147) 39.4% (85/216) Pisauridae Pisaura ancora 20.0% (1/5) 0% (0/1) 16.7% (1/6) Salticidae Plexippus selipes 28.6% (2/7) 0% (0/7) 14.3% (2/14) Pardosa pseudoannulata 16.7% (8/48) 10.0% (1/10) Lycosidae P.