of Acentria ephemerella larvae
Melanie Hempel & Elisabeth M. Gross
ABSTRACT: The larvae of the aquatic pyralid moth Acentria ephemerella (DENIS &
SCHIFFERMÜLLER) cause severe feeding damage on macrophytes in Lake Constance, esp. on Potamogeton spp. and Myriophyllum spicatum L. Earlier studies showed a better growth performance on tannin poor pondweeds than on tannin rich M. spicatum. In two independent experiments, we tested if the tannin degrading betaproteobacterium Matsuebacter sp. would enhance the growth of A. ephemerella larvae by detoxifying the plant secondary metabolites and by providing further carbon and nitrogen sources. First instar larvae were offered three differently treated M. spicatum plants: axenic M. spicatum (AX), axenic M. spicatum colonized with Matsuebacter sp. (MATS) and mesocosm M. spicatum (MESO). Fresh food was supplied every three to four days ad libidum when headcapsule width of the larvae was measured. The daily growth rates of larvae were 9 – 13 µm day-1 on axenic, 7 – 15 µm day-1 on Matsuebacter sp.-colonized plants and 5 – 12 µm day-1 on mesocosm plants. The mortality of larvae was highest in the MESO set-up (80%), while the other two set-ups had mortalities from 35 - 52% in both experiments. Treatments with AX and MATS had similar carbon and nitrogen contents [388 ± 6 mg C (g dm)-1 and 43 ± 2 mg N (g dm)-1] while the MESO treatment contained 228 ± 17 mg carbon (g dm)-1 and 12 ± 3 mg nitrogen (g dm)-1. Since no growth difference was found between axenic and Matsuebacter sp.-colonized plants, the bacteria were neither an additional nutrient source for the larvae, nor did they affect the larval growth by possibly modifying polyphenols. Thus, one single bacterial strain will most likely not effectively influence larval growth.
Keywords: Myriophyllum spicatum, herbivores, Lepidoptera, betaproteobacteria, biofilm.
INTRODUCTION
Bacteria are found in almost every environment where they cover the range from commensals to pathogens. In many organisms, the majority of bacteria is not pathogenic but rather commensal. Especially important are bacteria in the intestines.
In t has been shown for a variety of habitats that gut microbiota are essential for the successful processing of forage or to synthesize specific amino acids or sterols (Dillon
& Dillon 2004). Usually, the gut bacteria are adapted to certain environments and food sources. But not all forage can be equally well digested. Some diets contain plant secondary compounds that hamper the digestion by inhibiting the gut microbiota. Well known compounds in nature with this effect are tannins. They chelate iron, complex proteins and nutrients (Scalbert 1991), and decrease the nitrogen availability in ruminants (McSweeney, 1999).
Tannins are well recognized as plant allelochemicals in terrestrial and aquatic ecosystems. The hydrolysable tannins of the macrophyte Myriophyllum spicatum L.
(Haloragaceae) inhibit the photosystem II of cyanobacteria (Leu et al. 2002), and larvae of the aquatic moth Acentria ephemerella larvae (Pyralidae, DENIS &
SCHIFFERMÜLLER) grow slower on M. spicatum than on the pondweed Potamogeton perfoliatus, which does not contain hydrolysable tannins (Choi et al. 2002). Further, gut bacteria of A. ephemerella were inhibited by tannins from M. spicatum (Walenciak et al. 2002). Although in the past, herbivory on aquatic macrophytes was considered rare (Shelford 1918, Gregory 1983), high abundance of herbivores may have a substantial impact on macrophytes (Newman 1991). In Lake Constance, A. ephemerella larvae cause substantial damage to apical meristems of M. spicatum and P. perfoliatus. Larvae are usually found in densities higher than 0.8 individuals per shoot, which is considered to cause a decline of M. spicatum (Painter & McCabe 1988, Gross et al. 2002).
While tannin rich plants are considered to be well protected against herbivory (Feeny 1970), a tannin rich forage does not have to be detrimental. Rats fed with a diet rich in condensed tannins, select towards gut microbiota capable to degrade
tannins (Smith & Mackie 2004). Further, tannins in green tea, for example, act as prooxidants in yeasts because they reduce reactive oxygen species (Maeta et al. 2007).
Besides plant allelochemicals, plant stoichiometry is important for insect growth and food choice. Aquatic herbivores consuming elementally imbalanced food have a diminished conversion efficiency of ingested carbon into new biomass, and the gross growth efficiency is reduced (Elser et al. 2000). Food plants containing low amounts of phosphorus also impair growth and reproduction (Urabe & Watanabe 1992).
Lepidoptera prefer plants with the highest nitrogen content, which is also one of the major determinants of food quality (Newman 1991, White 1993).
Schultz and others (Schultz et al. 1992) assume that negative impacts of plant polyphenols on herbivores may be nothing more than a by-product of plant-microbe interactions. Thus, we asked whether A. ephemerella growth on tannin rich M. spicatum is promoted if the plant is colonized with a bacterial strain that degrades tannins. In 2005, Matsuebacter sp. was isolated in enrichment experiments from the surrounding water of M. spicatum. This betaproteobacterium is able to degrade tannic and gallic acid constitutively (Müller et al. 2007). To investigate the effect of this bacterium on the growth of freshly hatched A. ephemerella larvae, we also offered axenic (= bacteria-free) M. spicatum and M. spicatum grown in an outdoor mesocosm, which were colonized with a natural biofilm.
MATERIALS & METHODS
Larvae. Two egg clutches with 90 – 200 eggs were collected in July 2007 on Potamogeton perfoliatus. These egg clutches were reared in sterile filtered lake water (0.2 µm) at 20 ± 2 °C in with permanent aeration. After hatching, larvae were fed for seven days with axenic M. spicatum to avoid further bacterial contamination and to accustom the larvae to the food supplied in the assay. We performed one feeding experiment with each of the egg clutches.
Food sources/plant material. Three differently treated kinds of M. spicatum were offered during the experiment resulting in the three set-ups: axenic plants (AX), axenic plants colonized with the tannin degrading bacterium Matsuebacter sp.
(MATS) and mesocosm M. spicatum (MESO). Axenic plants were cultured as described in (Gross 2003) and supplied with fresh medium every two weeks to ensure the plants fed were of the same nutritious state. Myriophyllum spicatum was readily colonized within 48 h by an actively growing liquid culture of Matsuebacter sp. that was diluted to an optical density (OD600nm) of 0.2 in 100 ml sterile artificial tap water (DIN EN ISO 7346-3:1998-03). Previous studies proved that 48 h are sufficient to ensure colonization of the plant (Wicks, Hempel and Gross, submitted for publication). We used mesocosm M. spicatum to monitor the larval growth on plants with a natural biofilm. The mesocosm is a 2×2×1 m concrete basin in the yard of the Limnological Institute filled with Lake Constance sediment and water (flow rate ca.
20 l h-1), and densely planted with M. spicatum. The plants were exposed to the same environmental conditions as in the field and thus should be comparable to field plants. Mesocosm plants were collected 48 h before they were fed to the larvae. To provide comparable conditions for all plants compared to Matsuebacter sp.–colonized M. spicatum, axenic and mesocosm plants were also incubated for 48 h in sterile artificial tap water prior to feeding.
Bacterial cultures. Matsuebacter sp. cultures were grown at 20 ± 2 C° in medium B (Müller et al, 2007) without yeast and tryptone but with 3 mM succinate for permanent cultures, and 5 mM succinate for experiments. The bacteria were
harvested after 24 h hours in the exponential growth phase. The cells were washed twice with artificial tap water to remove Medium B and the cell suspension was adjusted to an OD600 of 0.2.
Feeding assays. Feeding assays were performed in sterile cell culture dishes. Each dish contained 4 ml artificial tap water, 3 – 4 leaves of the respective plant material and one larva. Larvae were distributed randomly to the three set-ups. Both experiments started when the larvae were in average seven days old and ended after 21 days. Every three to four days, the headcapsule width of the larvae was measured under the stereomicroscope with digital imaging, and fresh food and water were supplied in a new, sterile dish. Food was always supplied ad libidum. The experiments were conducted in a Sanyo MLR 350 environmental test chamber (SANYO Electric Biomedical Co., Ltd., Japan) at 20 °C and a light regime of 16 hours light (level 3; equal to 100 µmol photons m-2 s-1) and 8 hours darkness.
Chemistry. At each feeding day, a subsample of the plants of each treatment were analysed for total phenolic content, carbon and nitrogen (Choi et al. 2002). At the end of the experiments, all larvae were frozen and the intestines removed. The gut was transferred into sodium pyrophosphate (see below). The larval bodies and heads were dried at 60 °C and each 6 - 9 larvae were pooled and subjected to carbon and nitrogen measurements to obtain the nutrients accumulated during the experiments.
We removed the intestines, because we only wanted to analyze the accumulated nutrients in the body and no those present in the gut, which would reflect recent feeding rather than real nutrient uptake. To remove the intestines, the larval head was fixed with tweezers and the body was cut off behind the headcapsule with a scalpel without cutting off the intestines from the head. The head with the intestines was pulled out of the body, and the intestines cut off. Due to the small size of the larvae, all dissections were performed under a stereomicroscope.
Bacterial counts. At the end of the experiment, three guts of each treatment were resuspended in 0.9 ml 0.1 M sodium pyrophosphate (Na4P2O7×10 H2O) and fixed with 0.1 ml 37% formaldehyde for bacterial enumeration. The guts were transferred
to an ultrasonic bath (Laboson 200 ultrasonic bath, Bender & Hobein) for 60 s, shaken for 15 min (1100 rpm, Eppendorf Theromixer, 20 °C) and again exposed to 60 s ultrasonication (Walenciak 2004). The suspension was filtered onto polycarbonate filters (0.2 µm) and bacteria were stained with DAPI (4ʹ,6–Diamidino–2–phenylindol, 1 µg ml-1, 5 min). Stained cells were counted under an epifluorescence microscope (Labophot 2, Nikon) at an excitation wavelength of 549 nm. The bacterial cell counts were related to gut volume. We estimated the gut volume with a correlation for headcapsule width to gut length (100 µm headcapsule width equals 1.6 mm gut length). A mean gut diameter of one millimetre was assumed (Walenciak 2004).
Statistical analysis. We compared the different treatments with one–way ANOVAs. If the normality test failed despite transformation of the data, we used ANOVA on ranks with the Tukey post hoc test or Dunn´s method. The comparison of the food quality between the different experimental days was also performed with one–way ANOVA.
RESULTS
Experiment I. At the beginning of experiment 1 in July 2007 the headcapsule width of the 78 larvae was 173 ± 5 µm. The larvae were distributed to the three different treatments (Ax n = 27, MATS n = 26, MESO n = 25). In the set-up with Matsuebacter sp.–colonized plants and axenic plants, the larvae grew better than with mesocosm plants (Figure 1A). The average headcapsule growth rates were 15 ± 3 µm day-1 (mean ± 1SD for all data given), 13 ± 3 µm day-1 and 12 ± 2 µm day-1, respectively and did not differ significantly (P = 0.67). At the end of experiment I, the larvae in treatments MATS and AX had a headcapsule width of 496 ± 44 µm (n = 17) and 455 ± 67 µm (n = 16), respectively, while larvae fed with mesocosm plants had a headcapsule width of 448 ± 46 µm (n = 5; Figure 5.1A).
During the 21 days, several larvae died. The highest mortality was observed in the mesocosm treatment. Here, 80% of the larvae died during the experiment, while in the AX and MATS treatments 40% and 35% of the larvae died, respectively.
A)
100 200 300 400 500 600
B)
5 10 15 20 25 30
Headcapsuslse width [µm]
100 200 300 400 500 600
AX MATS MESO
Figure 5.1. Growth of Acentria ephemerella larvae on differently treated Myriophyllum spicatum leaves. A) Feeding experiment I B) Feeding experiment II with three differently treated M. spicatum.
AX axenic M. spicatum, MATS M. spicatum colonized with Matsuebacter sp., MESO mesocosm M. spicatum. Mean ± 1SD.
The total bacterial cell counts in the larval gut as determined by DAPI ranged from 4 ± 3×106 cells (mm gut volume)-3 to 8 ± 3×106 cells (mm gut volume)-3 with no distinct differences for the set–ups (Figure 5.2A, one–way ANOVA, F = 1.105, P = 0.39).
The carbon/nitrogen molar ratio (C/N) was 11 ± 1 and 10 ± 0.3 for axenic and Matsuebacter sp.–colonized plants, respectively (Table 5.2). Mesocosm plants had a C/N ratio of 21 ± 3 (Table 5.2). The total phenolic content was 60 ± 5 mg (g dm)-1 in axenic, 64 ± 5 mg (g dm)-1 in Matsuebacter sp.–colonized and 35 ± 11 mg (g dm)-1 in
0 during the experiment with axenic and Matsuebacter sp.-colonized plants, respectively. For each treatment only two measurements were possible since at least five larvae had to be pooled for one assay. Larvae fed mesocosm plants accumulated
Figure 5.2. Total bacterial cell counts in the gut of differently fed Acentria ephemerella larvae at the end of the experiment. A) Feeding experiment I B) Feeding experiment II. AX axenic Myriophyllum spicatum, MATS Matsuebacter sp. colonized-M. spicatum,
MESO mesocosm M. spicatum. n = 3, mean ± 1SD
242 mg C (g dm)-1 and 85 mg N (g dm)-1 in 21 days (Table 5.4). Due to the high mortality in some of the treatments, a higher replication was not possible.
Experiment II. In the second experiment, 132 larvae were distributed to the different treatments (AX n = 45, MATS n = 43, MESO n = 44) and had an averageheadcapsule width of 325 ± 27 mm. The daily growth rate was lower than in the first experiment.
Larvae fed axenic M. spicatum grew 9 ± 3 mm day-1, larvae fed Matsuebacter sp.–
colonized plants 7 ± 3 mm day-1 and larvae on mesocosm plants grew 5 ± 3 mm day-1. The daily growth rate of larvae fed axenic M. spicatum was significantly higher, than those fed mesocosm plants (ANOVA on ranks, P = 0.006). In this experiment, larvae fed axenic M. spicatum and Matsuebacter sp.–colonized M. spicatum grew 488 ± 58 µm and 466 ± 57 µm, respectively. The larvae grown on mesocosm plants had a final headcapsule width of 433 ± 52 µm (Figure 5.1B).
The mortality was 52% for larvae on axenic plants (n = 22), 35% for larvae on Matsuebacter sp.–colonized plants (n = 28) and 75% for larvae reared on mesocosm plants (n = 11).
Table 5.1. Statistical analysis for the comparison between the headcapsule widths during the experiment in the three set-ups by one–way ANOVA with Holm-Sidak post hoc test. On all other sampling days, but those displayed, larval headcapsule width was not significantly different. Ax:
axenic Myriophyllum spicatum, MATS: Matsuebacter sp.–colonized M. spicatum, MESO: mesocosm M. spicatum
a) ANOVA on ranks with Dunn´s method was used
Table 5.2. Chemical parameters of the differently treated Myriophyllum spicatum in Experiment I. Statistical analysis of data was performed with one-way ANOVA with α < 0.05, except for phosphorus and C/N, which were analyzed by ANOVA on ranks with a Tukey post hoc test. Statistically significant different groups after post-hoc tests are indicated by different groups. AX axenic Myriophyllum spicatum MATS Matsuebacter sp.-colonized M. spicatum MESO mesocosm M. spicatum
Table 3 Chemical parameters of the differently treated Myriophyllum spicatum in Experiment II. Statistical analysis of data was performed with one-way ANOVA and α < 0.05, except for carbon and C/N, which were analyzed by ANOVA on ranks with a Tukey post hoc test. Statistically significant different groups after post-hoc tests are indicated by different groups. AX axenic Myriophyllum spicatum MATS Matsuebacter sp.-colonized M. spicatum MESO mesocosm M.
spicatum
The total bacterial cell counts were slightly lower than in the first experiment.
Larvae fed with mesocosm plants had a slightly higher bacterial cell counts in the gut [7 ± 1×106cells (mm gut volume)-3] than the larval guts of the other two treatments, which were rather identical [Figure 5.2B, AX 5 ± 1 ×106cells (mm gut volume)-3, MATS 5 ± 0.8 ×106cells (mm gut volume)-3, one–way ANOVA, F = 2.309, P = 0.095].
The plant chemistry between both experiments was quite similar. In the second experiment the C/N ratio of axenic and Matsuebacter sp.–colonized plants was 10 ± 0.4 and 11 ± 0.5 respectively, and 24 ± 3 in mesocosm plants (Table 5.3). The total phenolic content during the experiment did not vary much (Table 5.3) with 61 ± 7 mg (g dm)-1 for axenic, 62 ± 3 mg (g dm)-1 for Matsuebacter sp.–colonized and 34 ± 6 mg (g dm)-1 for mesocosm plants. The phosphorus content was similar in axenic and Matsuebacter sp.–colonized plants [9 ± 2 and 9 ± 1 mg (g dm)-1, respectively] and 1 ± 0.3 mg (g dm)-1 in mesocosm plants. The food sources did not differ between the different feeding time points (ANOVA on ranks, P values were always > 0.6). In this experiment, too, the set-ups AX and MATS had rather identical plant chemistry (Table 5.3).
Carbon and nitrogen content in the larval bodies were assessed after the experiment ended at day 21. In this experiment, the larvae accumulated 486 – 511 mg C (g dm)-1 and 88 – 97 mg N (g dm)-1 in the set-ups AX and MATS. Larvae fed mesocosm plants had a nitrogen content of 110 mg (g dm)-1 and a carbon content of 467 mg (g dm)-1 (Table 5.4).
Table 5.4. Carbon and nitrogen content in larval bodies after 21 days on different diets. Six to nine larvae were pooled for each replicate. AX axenic Myriophyllum spicatum, MATS Matsuebacter sp.-colonized M. spicatum, MESO mesocosm M. spicatum, na no larvae were available for measurements
AX MATS MESO a)
a) Due to the high mortality in this treatment, no further replicates were possible.
DISCUSSION
We compared the growth performance of Acentria ephemerella larvae on differently treated Myriophyllum spicatum plants and were interested, if the tannin–degrading bacterium Matsuebacter sp. would enhance the growth of the larvae, which usually grow slower on tannin rich M. spicatum than on tannin poor Potamogeton perfoliatus (Choi et al. 2002).
The high mortality we observed in the treatment with mesocosm plants is probably due to the leaf toughness. The mesocosm plants were very stiff and covered with lime. Usually, larvae in this treatment did not show signs of food consumption (faecal pellets, coloured gut) in contrast to larvae fed axenic or Matsuebacter sp.-colonized plants. Those leaves were light green and soft, the larval guts were green coloured and we found faecal pellets frequently. Thus, we assume that larvae could not mechanically handle mesocosm plants due the high ash content. In another growth experiment, we offered lake-M. spicatum and it was well used by the larvae (data not shown). We further assessed the ash free dry mass (AFDM) of field and mesocosm M. spicatum and found lower AFDM values in the leaves of mesocosm plants (79% ± 10% and 53% ± 15%, respectively, Student’s t-test, P < 0.001), indicating a higher ash content. We assume that the higher anorganic content in mesocosm
without natural enemies. Probably the leaf toughness is an adaption to increased grazing pressure.
That the larval growth was rather identical on axenic and Matsuebacter sp.-colonized M. spicatum may be related to the overall better nutrient content of the plants and the lower ash content in comparison to mesocosms plants. The M.
spicatum plants for those set ups originated from a non limiting plant medium and the bacteria were obviously not an additional carbon or nitrogen source (Table 5.2 and 5.3). Since Lepidoptera are known to prefer food with a high nitrogen content (Newman 1991, White 1993), the higher larval growth rate on both axenic and Matsuebacter sp.-colonized plants could also be explained by the twice as high C/N ratio of mesocosm plants (Table 5.2 and 5.3). Further, mesocosm plants had a lower phosphorus content. The growth of mayflies and Manduca sexta larvae was also negatively affected at high C:P ratios (Frost & Elser 2002, Perkins et al. 2004). Thus, larvae that were able to feed on mesocosm M. spicatum had less nutritious food than in the other two assays and might even be P-limited. These data are also confirmed by the carbon and nitrogen content that accumulated in the larval bodies. In the AX and MATS treatments of experiment I, the carbon content of larval bodies was twice as high as in larvae fed the mesocosm plants. The nitrogen content in the larval bodies was similar between the three treatments with slightly higher values in larvae fed Matsuebacter sp.-colonized plants (Table 5.4). This can be explained by homeostasis of the larvae. Despite abundant food availability, invertebrates might grow slower because of nutrient deficiency of their food that has to be compensated (Sterner & Hessen 1994). Unfortunately due to the high mortality and necessity to pool many larvae for one C/N measurement, we do not have enough replicates to perform statistical analysis and confirm these data. We also had not enough larvae to perform analyses of larval P content. Thus the C/N values can only be used as indicators for possible limitations and storage mechanisms. In the second experiment the carbon content was rather similar between all treatments while the accumulated nitrogen in the larval bodies was rather similar in all food sources.
Besides measuring the headcapsule width and nutrient content of plants and larvae, we also assessed the bacterial numbers in the gut. Interestingly, the bacterial numbers in the gut were equal between different set ups. This is further evidence that bacteria are inherited in early larval stages and also that bacteria pass rather quickly through the gut (Dillon & Dillon 2004). We expected that larvae fed mesocosm and Matsuebacter sp.-colonized plants would inherit more bacteria than those fed axenic plants. One reason for equal bacterial numbers could arise from the rearing of the larvae. As long as larvae were hatching, the Potamogeton perfoliatus leaf, which had a natural bacterial biofilm, and on which the egg clutch was found, was left in the Erlenmeyer flask and only removed if all larvae were hatched. Usually, hatching took three days in which also axenic M. spicatum was offered, but newly
Besides measuring the headcapsule width and nutrient content of plants and larvae, we also assessed the bacterial numbers in the gut. Interestingly, the bacterial numbers in the gut were equal between different set ups. This is further evidence that bacteria are inherited in early larval stages and also that bacteria pass rather quickly through the gut (Dillon & Dillon 2004). We expected that larvae fed mesocosm and Matsuebacter sp.-colonized plants would inherit more bacteria than those fed axenic plants. One reason for equal bacterial numbers could arise from the rearing of the larvae. As long as larvae were hatching, the Potamogeton perfoliatus leaf, which had a natural bacterial biofilm, and on which the egg clutch was found, was left in the Erlenmeyer flask and only removed if all larvae were hatched. Usually, hatching took three days in which also axenic M. spicatum was offered, but newly