What is the functional role of plant-associated invertebrate diversity in submerged
vegetation?
Author: John Onita
1. Introduction and Literature Review
The fact that biomass, species composition and seasonal duration of aquatic vegetation
determine the composition and abundance of macroinvertebrates, have long been established
(Lodge, 1985; Rooke, 1986; Kornijow, 1989). But one thing that is not yet clear enough is,
understanding the degree to which plant associated macroinvertebrates play key roles in
regulating the dynamics of micro algal biomass in fresh water vegetation. Several studies have
reported the fundamental importance of grazers in maintaining the health of some specific
aquatic vegetation such as seagrass meadow (Nickles et al., 1993; Jernakoff and Nielsen, 1997).
More so, the regulation of abundance and growth of epiphytic algae which competes for light
availablility with aquatic vegetation by invertebrate grazers have been reported (Montfrans et
al., 1984). It is also very important to mention that earlier studies have shown the modification
effects of invertebrates grazing on periphyton and epiphyton composition (Russo, 1989) which
in turn provides a connection in the aquatic food web between primary producers and higher
trophic levels (Orth, 1992).
In contrast to most reports on the regulatory functions of grazing on abundance and density of
microalgal species, some recent studies indicates that invertebrate grazing might not have the
most reported potential to reducing microalgal abundance in highly eutophic lakes (Lotze and
Worm, 2002; Lapointe et al., 2004). This conclusion was based on the explanation that
eutrophication modifies the diversity, species richness and species composition of plant
communities (Korpinen et al., 2007). Another explanation to the conclusion was that
invertebrate grazing effects on plant communities rely on productivity (Hillebrand et al., 2000),
even as it was reported that in unproductive lakes, plant species richness was decreased by
grazing pressure, but when compared to productive lakes, grazing suppressed dominant algae,
hence an increase in algal diversity and species richness (Korpinen et al., 2007). However,
Hillebrand et al.,(2000) shed more light on this conclusion by reporting that grazing increased
microalgal diversity and species richness only in productive conditions especially at the ambient
nutrient level where grazing impact was negative on both diversity and species richness.
Possible reasons given to explain the difference in grazing effects of invertebrate species on
microalgae was the probable preferences of grazers to a variety of microalgal species (Jernakoff
and Nielsen, 1997). This is because little knowledge exist about the difference in functional roles
of invertebrate species in controlling macroalgal growth, for example, comparing a species
functional roles with another as in the case of gastropod and amphipod species respectively
(Jernakoff and Nielsen, 1997).
It is however, very important to remind our readers that studies on mesograzers, vis-a-vis
herbivory on algae species have predominantly centered on examining the roles of eukaryotic
algae as food and habitat for marine grazers (as viewed by Paul et al., 2001). A clear-cut reason
for this view was given by Cruz-Rivera (2006) who further explained that many community
studies which described algae species in fresh water systems have concentrated particularly, on
explaining the factors underlying the control of algal blooms because it is usually linked to
eutrophication, disturbance and anthropogenic habitat degradation, hence the continuous
existence of algae in aquatic ecosystems is seen as “abnormal” instead of normal component of
the community.
In tune with the view expressed above, feeding preferences of mesograzers in relation to
macroalgal species have been widely reported (Cornwell et al., 2009). For example, the study of
the large molluscan grazer – Abalone, feeding on macroalgae species of marine origin indicates
that species of Abalone from the northern hemisphere prefer the brown algae species as their
feeding habits (Guzman del Proo et al., 2003), while their counterparts from South-East Asia
tend to feed on red algae species (Tahil and Junio Menez, 1999).
More so, some studies have suggested that benthic blue-green algae species from tropical
regions, could play great ecological roles by serving as sources of food and habitat to marine
meso-grazers (Cruz-Rivera and Paul, 2002, Paul et al., 2001). For instance, these studies
described the feeding preferences of the small sea hare Stylocheilus striatus and concluded that
this organism; which lives in Marine blue-green algae mats, could feed preferentially on the
blue-green algae species of the genus Lyngbya, at the same time that the organism has the
choice of other algae species, particularly macroalgae and cyanobacteria species as food
sources.
Given the stated claims of varying tendencies among mesograzers with regards to their
preferences of algae species as food sources, it is reasonably important to suggest that
mesograzers has the potential to feed on alternative food sources other than algae species.
Supporting this hypothesis, several studies have shown that some aquatic invertebrates feed on
a variety of food sources (Dangles, 2002; Mihuc and Mihuc, 1995; Friberg and Jacobsen, 1994).
For example, Hirabayashi and Wotton, (1999) reported that chironomid larvae has the ability to
feed on many food types including algae, detritus and associated microorganisms, macrophytes,
and woody debris.
Similarly, Baker et al., (2010) conducted a laboratory study to quantify the rates of consumption
of native and non-indigenous aquatic plants in Florida using the snail Pomacea insularum
(Gastropoda: Ampillariidae). The authors concluded that more than 50% of the plants were
consumed by the snail. In the same vein, study showing the efficient consumption rates of
decayed Alnus glutinosa leaves by Asellus aquaticus have long been reported (Adcock, 1982);
even as another study showed the feeding ability of the decapod Cambous bartonii on leaf litter
(Huryn and Wallace, 1987). More recently, Callisto et al., (2007) conducted an investigation to
show the possible use of head water stream leaf litter as food source for chironomids and
concluded that some chironomids can use leaf litter of riparian vegetation as a complementary
food source.
Therefore, with the feeding abilities of mesograzers on algae species and frequent
diversification of mesograzers’ feeding potential to alternative feeding sources (e.g. dead leaves)
extensively reviewed, it is surprising to note that to-date, no studies have compared the relative
use of blue-green algae as possible food source among different mesograzer species in fresh
water systems (Cruz-Rivera and Paul, 2006), hence there were two general aims of this study;
the first was to compare the use of epiphyton as food source among mesograzer species. The
second was to compare the use of dead leaves as alternative food source among mesograzer
species.
Hypotheses
(a) – Mesograzer species under controlled laboratory conditions, will consume epiphyton
biomass at the same grazing rates.
(b) – Mesograzer species feeding rates on epiphyton biomass is proportional to the rate of
feeding on dead leaves.
2. Materials and Methods
Study site and experimental species.
Two types of experimental approach were adopted during the course of the investigation. The
experimental procedures were all conducted in the laboratory.
(i) Epiphyton culture in the laboratory:
Epiphyton was cultured under a controlled experimental conditions in a Green House located in
the Department of Biology, Linköping University, Sweden. This process lasted for five months
between March – July. During the experiment, two transparent plastic tanks measuring 3 feets
(length), 2.5 feets (width) and 2 feets (height), were used. Pond water from the Linköping
University wetlands was used through out the duration of the experiment simply to maintain
some degree of natural water chemistry. Pond water was filtered using a standard plankton
sampling net of mesh size 20 µ and a plastic funnel of 17-20 cm diameter and used to fill up to
halve the capacity of the culture tanks. This was to control the unwanted addition of algal
biomass to the culture as well as remove zooplankton grazing effects on the culture.
Temperature was kept constant at 15 ºC in the green house. Also, there was a constant light
supply directly on to the culture tanks , a technique designed to stimulate algal growth after the
natural process of photosynthesis where radiant energy from the sun drives the system. A
wooden stage was constructed to fit into the bottom layer of each of the plastic water tanks
which were partitioned into 3 rows of two equal parts facing each other in opposite direction. In
all, each tank had 6 rows of partitioned wooden stage supported with heavy stone weights
(approximately 16 kg per tank) which purpose was to keep the wooden stage constantly sank to
the bottom of the tanks even when the wooden parts completely absorbs water and tend to
float. White tiles carefully cut into a specified size of 12 cm in length and 9 cm in width were
placed vertically on to the wooden stage rows in the tanks. Each row contained 5 pieces of
white tiles , bringing to a total of 30 tiles per tank (i.e. 5 tiles × 6 rows in each tank). Macro algal
species of un-identified samples were collected from the Linköping University wetland ponds
and carefully introduced into the culture tanks using a plastic petri-dish. There was a constant
source of aeration in the culture tanks through out the experimental period. Using a standard
measuring cylinder, 12.5 ml of diluted nutrient solution was added to each tank once every
month to maintain a uniform algal growth firmly attached to the tiles.
Micro algal species were identified and counted under a dissecting microscope. For the process
of algal identification, samples of algae culture from the two tanks were taken separately and
placed in a 2 ml BOD bottles. 12 drops of lugol iodine solution were carefully added to each
bottle and labelled “A” and “B” denoting the respective culture tanks the samples were taken
and stored in a dark place.
However, in order to ascertain the grazing potential of invertebrate species on epiphyton, it was
necessary to diversify our investigation to cover the use of dead leaves as alternative food
source to invertebrates. Reason for this consideration was based on the idea that it would be
better to have a comparison of the expected grazing potential of invertebrates feeding on both
epiphyton and dead leaves in order to draw a meaningful conclusion.
(ii) (a) Mesograzers feeding on dead leaves in the laboratory
To investigate the grazing potential of aquatic invertebrates on alternative food sources other
than epiphyton, dead leave specimens were used to test this hypothesis. Indeed, the dead leave
samples were collected from natural leaf falls in the University wetlands.
Invertebrate species used in the feeding trials were collected in April – May 2009 and
acclimatized to laboratory conditions for 1-3 days before the actual experiment started.
Invertebrate species used for the experiment were: Lymnea stagnalis, Asellus aquaticus, and
Gammarus pulex respectively. Lymnea sp was sampled at Lake Tåkern, while Asellus and
Gammarus species were sampled at the University wetlands.
Three sets of experimental trials were adopted which involved single species and combination of
species exposed to dead leaf samlpes. There were 20 replicates per experimental trial and also
20 replicates of a controlled experiment for each trial. A typical illustration of how the feeding
trials were organized is show in appendix 1.
For the single species experimental set up, the number of individual species exposed per
replicate was one. Similarly, the number of individuals exposed in the two species combinations
were two, where each species was represented by one individual combined with the other
species’ representative per replicate. The third experimental set had all the three species
combined with each species also being represented by one individual per replicate respectively.
There was no aeration during the feeding trials.
Food:
Dead leaves were provided to invertebrates in form of conditioned leaves. Conditioning of
leaves means microbial conditioning of leaves for a period of 2 weeks in a laboratory aquarium
using a mixture of stream collected leaves as an inoculum and under strong aeration
(approximately 20g of leaves in a plastic bowl). The leaf types were identified as…….????.
Decaying leaves (senescent) were collected with nets, air-dried and stored dry until needed.
Consumption:
Dead leaves were provided to test species in the form of leaf discs. These discs were cut in pairs
from contiguous areas of the same leaf with an improvised mechanism which involved the used
of small metallic pipe of 2 cm diameter circumference and 6 inches in length. Using a wooden
piece, pressure was applied on the metallic pipe in a form of knock at the top end of the pipe
while the oval base of the pipe was carefully placed on a conditioned leaf sample folded into a
pair and placed on a wooden plat-form. This produced a perfect leaf disc- pair which was
separated with the aid of a forceps. One of the discs was exposed to each 20 replicate for each
species, while the other disc was placed in a Petri-dish with no invertebrate (control, 20 in
number for each species). This implies that for each species, there were 20 replicates of small
Petri-dishes with leaf disc exposed to a test species and also 20 replicates of leaf disc not
exposed to test species (control). Both the exposed and unexposed leaf-discs were numbered 120 with each number complimenting the other.
Each invertebrate species was allowed to feed for a minimum of 3 days. Before the exposure of
test species to the leaf-disc, specific and mean weights of test species where taken for both the
single and combined experimental trials respectively. Consumption was calculated as the
difference in mass between unexposed and exposed discs divided by the elapsed time in days
(i.e. 3 days). More so, weights of each exposed and unexposed leaf-discs were taken as wet and
dry mass. Values were expressed as ash free dry mass; where discs were dried at 50 C for 2
days in an oven, weighed, ashed at 500 C for 1 hour, 30 minutes and weighed again. A key issue
was the assessment of the observed consumption of exposed leaf-discs by test species as “well
eaten” and “slightly eaten” in order to enhance the ash mass determination process. In doing
this, there was need to invent a technique which took into consideration the fact that some leafdisc replicates of the exposed trials were completely consumed by test species leaving only tiny
remains that had the capability of possing problems during the ash determination process. We
resolved this problem by formulating a key which involved creating groupings for all the “well
eaten” and “slightly eaten” disc replicates across the different experimental trials to correspond
to similar groups carefully created for their control replicates (see appendix 1). In each group, a
calculated value in percentage was given to every replicate number constituting the group,
where a mean value of each group was calculated by taking the average weight of the leaf-disc
remains after exposure from respective group replicates (dry mass in grams), dividing by the
total group number and multiplying by 100. Similar procedure was also adopted to determine
the percentage ash value for the control replicates of each group.
There were a total of 28 groups consisting of both the replicate and control groups.
Conclusion of feeding taking place was done when the mass of exposed discs was significantly
lower (pair t-test, p 0.05) than the corresponding control disc as described by Friberg and
Jacobsen (1994); Graca et al., (2001).
(ii) (b) Mesograzers feeding on epiphyton in the laboratory
With a clear intention to compare the feeding rates of invertebrate species on both epiphyton
and dead leaves, it is expected that similar experimental methods be adopted for both
experimental scenarios. Indeed, much of the methodologies described in the dead leaves
experiment were adopted. To some extent, contrasting differences in methods did arise which
included the following:
-
There were no combinations of species during the epiphyton experiment as was tested
in the dead leaf experiment. Reason being that the sensitivity of epiphyton response to
experimental manipulations was too fragile to handle when compared to that of the
dead leaves. For example, the physico-chemical requirements of epiphyton in water
(e.g. temperature, dissolved oxygen etc) required a lot of carefulness and is better
controlled when observing a single species response to epiphyton feeding.
-
There was constant aeration of all the mini aquaria used in the epiphyton experiment
which was not the case in the dead leaf experiment. Reason being the simple difference
in the nature of the food sources involved in both experiments. For the dead leaf
experiment, the leaves were dead and were already in their organic state of decay,
where as, the epiphyton or micro algae used in the epiphyton experiment were living
organisms, hence needed the same quantity of dissolved oxygen required for their usual
metabolic activities in their natural environments to be available for their optimal
survival during the experimental trials.
-
The choice of a constant light source during the epiphyton experiment was a necessity
to provide energy required to maintain survival and continuous growth of algal biomass,
where as it was a matter of choice during the dead leaf experiment.
15 transparent rectangular plastic bowls measuring 44 cm in length, 32 cm in width and 17 cm in
height were used to store filtered pond water that were halve filled the capacity of each bowl.
Water filtration procedure was performed after the method described in the “epiphyton
culture” section above. Each bowl contained 3 mini aquaria measuring 17 cm in length, 14 cm in
width and 14 cm in height. For each mini aquarium, rectangular pieces of plankton net of mesh
size 20 µ were glued on to rectangular openings created on both sides, length of the mini
aquarium. This construction was possible with the aid of an electric heating cutter which was
used to carefully cut a rectangular shape of 10 cm length and 7 cm width on both sides, length
of the mini aquarium. The gluing process was performed using also an electric heating device to
apply melted glue on to the rectangular edges of the plankton net pieces and then placing it
immediately on the mini aquarium opening sides, applying pressure with hand and allowing to
solidify on cooling. In all, a total of 45 mini aquaria were used in the experiment (i.e. 3 mini
aquarium × 15 plastic bowls). More so, in the very centre of each mini aquarium, a transparent
plastic petri-dish of 5 cm base diameter and 6 cm top diameter was glued. Each petri-dish was
slashed open from the top middle half in a vertical position, down to a 1 cm distance from the
bottom of the dish, creating an inclined resting platform on which small tile pieces were firmly
placed in each mini aquarium. The slashed petri-dishes were also 45 in number.
45 squared pieces of white tiles with diameter 2 inches × 2 inches were careful cut with the aid
of a glass cutter. Another set of smaller transparent plastic Petri-dishes with diameter 2.5 cm
base, and 3.5 cm top diameter were carefully re-constructed to create an enclosure designed to
trap every mass of epiphyton during the experiment. These smaller Petri-dishes were cut open
entirely at usually closed bottom using the same electric cutter device earlier mentioned. The
bottom side was covered with glued circular plankton net of mesh size 20 µ cutted to fit the
actual diameter of the bottom side with a slight opening on each glued net to enhance air
bubble escape.
[Diagram showing apparatus here]
Epiphyton harvest from culture experiment.
Cultured macro algal biomass still firmly attached to white tile pieces substrates were carefully
harvested by hand-pulling each colonized tile from the culture plastic tanks and using a spatula
to gently scrape the tile surfaces off substantial quantity of macro algal biomass. One mini
plastic aquarium of the size 17 cm in length, 14 cm in width and 14 cm in height was used to
collect the algal biomass before exposure to invertebrate feeding.
Plankton net pieces of the mesh size 20 µ was cut in a measured size that doubled the size of
the mini aquarium used to store the algal biomass as described above. This net pieces was used
to cover the open surface of the mini aquarium while using 2 strings of rubber binds to tightly
tie the net round the rectangular shaped mini aquarium. Pressure was applied right in the
middle of the net surface by hand, to create a depression needed to help accelerate the algal
biomass quantity collection while gradually draining retained water molecules out and dropping
into the open mini aquarium space. The harvested algal biomass was allowed to drain off water
for 1 hour.
Epiphyton and invertebrate measurements.
With the aid of a stainless steel spatula, harvested algal biomass was collected and weighed to
firstly; a considered base-line weight for the experiment which was 3.0 g (blotted wet weight to
the nearest 0.01 g). This value was chosen to allow for easy observation of the rate at which the
invertebrates fed on the algal biomass and also to see if there could be a significant growth of
algal biomass simultaneously, while the experiment was in progress especially, in the controlled
experimental replicates where there was no grazing. More so, the value was suitable for the
experimental capacity in terms of space in the small constructed Petri dish environment in
which both the measured algal biomass and invertebrates were introduced. The base-line
epiphyton biomass of 3.0 g was the controlled experiment and there were 5 replicates for each
invertebrate species used in the experiment.
Further more, 3.0 g algal biomass (blotted wet weight to the nearest 0.01 g) was carefully
measured following the procedure described above and was transferred on to the rough
surfaces of each labelled small white tiles with a diameter of 2 inches × 2 inches. There were 45
of these tiles with the measured base-line 3.0 g algal biomass for both the exposed and
unexposed (controlled) experiments.
Invertebrate species used in the experiment were same as those used in the dead leaves
experiment. They were: Lymnea stagnalis, Asellus aquaticus, and Gammarus pulex respectively.
Two stocking densities of these invertebrates were considered and implemented. The first
invertebrate density was 5 individuals per replicate and was denoted as high density population,
while the second density was 3 individuals per replicate and was denoted as low density
population. For each species and density, there were 5 replicates. The invertebrates were
sampled after the procedure earlier described in the dead leaves experimental methods. They
were allowed to aclimatised to laboratory conditions for 2 weeks under continuous aeration.
For each invertebrate species and replicate, the initial weights of the total population were
taken before and after exposure to epiphyton biomass. For Lymnea stagnalis, a step further was
taken to ensure that two different weights were taken for each replicate population. This
involved firstly, taking weights of each population considering the mass of the animal muscles
together with the mass of their shells and secondly, weights of each population without shell.
The second option was possible by ensuring that each animal population was frozen to death in
the freezing room, and subsequent extraction of each animal muscle from the shell using an
office pin.
Food
Algal biomass of 3.0 g (blotted wet weight to the nearest 0.01 g) base-line weight was provided
to two distinct populations of invertebrate species. While still placed on the rough surfaces of
squared (2×2 inches) small white tiles after measurement, species densities were stocked right
on the tiles with measured algal biomass and smaller transparent plastic Petri-dishes (diameter
2.5 cm base, and 3.5 cm top diameter) with glued nets on one open end were used to cover
each stocked replicate. Getting the side of the Petri dish with nets facing up, a string of rubber
bind was used to bind the corked Petri dish on to each stocked tile and carefully placed to rest
vertically on the glued vertical platform in each mini aquarium. This process lasted for 1 minute.
Invertebrates were allowed to feed on algal biomass for 4 days. Aeration was constantly
supplied in each mini aquarium containing every stocked species density. Temperature in the
Green House where the experiment was set up was kept constant at 15 ºC. There was constant
light supply directly on each experimental block which consisted of 3 mini aquaria containing
two distinctly stocked species densities and a control, all in a larger plastic bowl.
[Diagram showing apparatus here]
Consumption:
With invertebrate species densities of 5 individuals per replicate (high density) and 3 individuals
per replicate (low density) exposed to each 3.0 g algal biomass (blotted wet weight to the
nearest 0.01 g) (base-line measurement) and a control (unexposed) 3.0 g algal biomass (blotted
wet weight to the nearest 0.01 g) for each species, grazing was allowed to take place for 4 days.
Weights of algal biomass was taken as “wet and dry” weights respectively. The “wet” weight
was taken before and after the experimental period of 4 days for each exposed and unexposed
algal biomass replicates, while the “dry” weight was taken only after the experimental period
for each exposed and unexposed algal biomass replicates.
At the end of the 4-day experimental period, invertebrate species in each exposed replicate
were carefully removed one-by-one using a forceps and transferred to a separately marked
Petri-dish. Both exposed and unexposed algal biomass in each replicate was carefully gathered
in the corked Petri dish confinement which did not permit any possible escape of algal biomass
into the open aquarium and allowed to drain off water for 1 hour. Using a stainless spatula and
forceps, each exposed and unexposed algal biomass replicate was removed from the corked
Petri dish and weighed while still wet. There-after, each wet-weighed exposed and unexposed
algal biomass replicate was carefully marked and transferred into a drying machine where the
replicates dried for 2 days at 50 C. Following the 2-day drying process, the dry weights of each
exposed and unexposed algal biomass replicate was taken.
Consumption (grazing) was calculated as the difference in mass between unexposed and
exposed algal biomass replicates, divided by the elapsed time in days. Conclusion of feeding
taking place was done when the mass of exposed algal biomass was significantly lower (pair ttest, p 0.05) than the corresponding control (unexposed) as was in the case in the dead leaves
experiment (Friberg and Jacobsen, 1994; Graca et al., 2001).
(iii) Data Analysis:
For the epiphyton experiment, the amount of algae (both in wet and dry weights) consumed by
each grazer species was calculated by using the formula below as cited in Cornwell et al., (2009).
F = [F.sub.1] ([F.sub.2] [+ or -] C) (cited in Cornwall et al., 2009)
Where:
F = actual food consumption in each given treatment
[F.sub.1] = amount of food offered, (3.0g)
[F.sub.2] = remaining amount of food at the end of 4 days trials
C = change in control algae without grazer species.
The change in weight of treatment (exposed) and control (unexposed) algae were determined at
the same time, and the control algae biomass were paired with the treatment algae biomass to
determine F for that particular trial (Cornwell et al., 2009).
Further more; to test for significant differences between grazer treatments in both the
epiphyton and dead leaf experiments, One-Way ANOVAs were performed using the factor
grazer composition and response variables micro algal initial weight (3.0g) and was followed by
Newman-Keuls post hoc test (composition effect) as recommended by Jaschinski et al., (2009).
For the dead leaf experiment; to detect significant grazer species richness effects, measures
comparing the three-grazer treatment against all single-grazer treatment (richness effect) were
adopted (Jaschinski et al., 2009). Further, Net biodiversity effects (∆Y) were calculated as
described by Loreau and Hector (2001) in other to further estimate the diversity effects. In doing
this, ∆Y was tested against zero with a paired t-test.
To estimate the expected effect of each grazer species in the combinations (Lymnaea + Asellus;
Lymnaea + Gammarus; Asellus + Gammarus), means of the single-grazer treatments (n = 20).
The expected increase in net biodiversity effects from the two to three species combinations
were tested with a linear regression analysis (Jaschinski et al., 2009).
3. Results
Mesograzers epiphyton feeding experiment.
Based on three different parameters of measurement (Actual food consumption, Food
consumption at varying densities, and Weight-specific consumption); a total of 18 paired t-test
of independent samples conducted for each pair of the three test species revealed 12 significant
differences (p < 0.05) in the quantity of algae consumed between the two species in each pair
(Table 1).
6 none significant differences occured in the same statistical test taking into account, the same
three parameters of measurement stipulated above (Table 1). On the other hand, an ANOVA
test conducted on the three test species at once for each of the three parameters of
measurement, revealed a highly significant differences (p < 0.000) in the amount of algae
consumed between the three species in all 6 test runs conducted (Table 2).
Actual food consumption (F); paired t-test
Measurement of the actual food consumption (F) over the 4-day period of experiment indicated
that Lymneae stagnalis showed a distinct feeding potential on the amount of algae consumed
both at high and low densities respectively (Fig. 1).
At high density (5 individuals per replicate), the pair of Lymneae stagnalis and Asellus aquaticus
showed a significant grazing effect on algal biomass (p < 0.033). More significant effects of
grazer species feeding abilities on algal biomass were demonstrated (Table 1). The grazing
impact of the pair of Lymneae stagnalis and Gammarus pulex was significant at p < 0.016 which
differed significantly from the pair of Lymneae and Asellus (p < 0.033). Indeed, both pairs
posseses positive values but the pair of Lymneae and Gammarus seem to show more significant
grazing impact on epiphyton biomass than the pair of Lymneae and Asellus (Table 1). There was
no significant grazing effect on algal biomass by the pair of Asellus aquaticus and Gammarus
pulex. Their impact was negative at p > 0.606 (Table 1).
At low density (3 individuals per replicate), the pair of Lymneae stagnalis and Asellus aquaticus
showed a greater grazing impact on agal biomass with a significant effect at
p < 0.001 than the pair of Lymneae and Gammarus, also with a significant effect at
p < 0.002. Both pairs demonstrated a positive grazing impacts by reducing epiphyton biomass
significantly. More so, there was no significant grazing effect on epiphyton biomass at low
density by the pair of Asellus and Gammarus with p > 0.855 (Table 1).
a
b
Fig I. Effect of grazing on epiphyton biomass during a 4-day feeding trial. Actual food consumption; (a) High density treatment (5
individuals per replicate); (b) Low density treatment (3 individuals per replicate). [1 – 3 on x-axis], (1) Lymneae stagnalis ;( 2) Asellus
aquaticus ;( 3) Gammarus pulex.
Food consumption at varying densities (FC); paired t-test
Measurement of food consumption at varying densities (FC) for the different pairs of grazer
species (see appendix 1 for details) indicated that at both high and low densities, the grazing
potential of Lymneae stagnalis on algal biomass is not questionable (Fig. 2).
a
b
Fig 2. Effect of grazing on epiphyton biomass during a 4-day feeding trial. Food consumption at varying densities; (a) High density
treatment (5 individuals per replicate); (b) Low density treatment (3 individuals per replicate). [1 – 3 on X-axis], (1) Lymneae
stagnalis ;( 2) Asellus aquaticus ;( 3) Gammarus pulex.
At high and low densities, the pair of Lymneae and Asellus; Lymneae and Gammarus both
showed significant grazing impacts on algal biomass at the same level of significance (p < 0.003)
(Table 1). The pair of Asellus and Gammarus continued to show no significant impact on algal
biomass both at high and low densities respectively. At high density, the pair was not significant
at p > 0.288 and at low density (p > 0.784) (Table 1).
Weight-specific consumption (WSC); paired t-test
Measurement of the weight-specific consumption (WSC) of the three grazer species indicated
that Gammarus pulex exhibited greater grazing potential on the quantity of algal biomass
consumed at both high and low densities than the other two test species (Fig. 3).
At high density, the pair of Lymneae and Gammarus reduced algal biomass significantly at p <
0.007. The same positive grazing impact on algal biomass was also displayed by the pair of
Lymneae and Asellus at p < 0.012. There was no significant grazing impact exhibited by the pair
of Asellus and Gammarus at p > 0.179 (Table 1). At low density, the pair of Lymneae and
Gammarus maintained their leading grazing effect on algal biomass with a significant impact at
p < 0.009. This positive grazing effect was followed by the pair of Lymneae and Asellus with a
significant value of p < 0.029. Again, there was no significant grazing impact on algal biomass by
the pair of Asellus and Gammarus with
p > 0.834 at low density (Table 1).
ANOVA test for epiphyton experiment
A one-way ANOVA test conducted to shed more light on the paired t-test analysis, detected a
highly significant difference (p < 0.000) among all three test species for all the three parameters
at all population types tested in the course of the experiment. Even with varying figures in
Mean, Standard Deviation and F-factors, the p-values of the species for all parameters tested
remained highly significant (p < 0.000) (Table 2).
Figures 1-3 explained the meaning of this results by showing the grazing impacts of respective
test species on algal biomass at individual scale, instead of at paired level of significance as was
tested by the paired t-test analysis. This implies that each test species had a significant grazing
effect on epiphyton biomass; taking into account the various parameters of measurement as
indicated by the difference in Mean and Standard Deviation values for the respective mesograzer species tested (Table2).
a
b
Fig 3. Effect of grazing on epiphyton biomass during a 4-day feeding trial. Weight – specific consumption; (a) High density treatment
(5 individuals per replicate); (b) Low density treatment (3 individuals per replicate). [1 – 3 on X-axis], (1) Lymneae stagnalis ;( 2)
Asellus aquaticus ;( 3) Gammarus pulex.
Table I. Statistical summary of paired t-test analysis of test species (Lymneae, Asellus and Gammarus)
________________________________________________________________________________________________________
Epiphyton experiment data analysis
________________________________________________________________________________________________________
Parameter of
Measurement
Population type
Paired t-test analysed
Mean
Standard Deviation
Condition; t(5)
p – Value
Inference
________________________________________________________________________________________________________
Actual Food
Consumption
(g)
High Density
• Lymneae + Asellus
0.69600
0.48671
3.198
0.033
Significant
• Lymneae + Gammarus
0.73760
0.41052
4.018
0.016
Significant
• Asellus + Gammarus
0.04160
0.16660
0.558
0.606
Non Significant
_________________________________________________________________________________________________________________
Low Density
• Lymneae + Asellus
• Lymneae + Gammarus
• Asellus + Gammarus
1.21900
1.24000
0.02100
0.30156
0.38238
0.24095
9.039
7.251
0.195
0.001
0.002
0.855
Significant
Significant
Non Significant
_________________________________________________________________________________________________________
Food Consumption High Density
• Lymneae + Asellus
0.32700
0.11087
6.595
0.003
Significant
at varying
(5 individuals
• Lymneae + Gammarus
0.32280
0.11132
6.484
0.003
Significant
densities
per replicate)
• Asellus + Gammarus
-0.00420
0.00766
-1.226
0.288
Non Significant
(g)
___________________________________________________________________________________________________________________
Low Density
(3 individuals
per replicate)
• Lymneae + Asellus
• Lymneae + Gammarus
• Asellus + Gammarus
0.32800
0.32720
-0.00080
0.07003
0.07387
0.00610
10.473
9.904
-0.293
0.000
0.001
0.784
Significant
Significant
Non Significant
_________________________________________________________________________________________________________
Weight – Specific
Consumption
(g)
• Lymneae + Asellus
-3.99760
2.03177
- 4.400
0.012
Significant
• Lymneae + Gammarus
-2.73340
1.21096
-5.047
0.007
Significant
• Asellus + Gammarus
1.26420
1.73838
1.626
0.179
Non Significant
___________________________________________________________________________________________________________________
High Density
Low Density
• Lymneae + Asellus
• Lymneae + Gammarus
• Asellus + Gammarus
-2.79820
-2.51220
0.28600
1.88033
1.17496
3.03503
-3.328
- 4.781
0.211
0.029
0.009
0.843
Significant
Significant
Non Significant
__________________________________________________________________________________________________________
Table I. Statistical summary of paired t-test analysis of test species (Lymneae, Asellus and Gammarus).......continuation...
___________________________________________________________________________________________________________
Dead Leave experiment data analysis
____________________________________________________________________________________________________________
Parameter of
measurement
Population type
*Paired t-test analysed
Mean
Standard Deviation
Condition; t(5)
p – Value
Inference
____________________________________________________________________________________________________________
Consumption Rate
(CR)
(g)
Single species
treatment
• Lymneae + Asellus
• Lymneae + Gammarus
• Asellus + Gammarus
0.01838
0.00680
-0.01157
0.01950
0.01998
0.01166
4.215
1.522
- 4.439
0.000
0.144
0.000
Significant
Non Significant
Significant
________________________________________________________________________________________________________________________________________
Replicate total
dry mass (RTD)
(g)
Replicate actual
ash value (RAA)
(g)
Combined species
treatment
(Group 1-28)
Combined species
treatment
(Group 1-28)
*Regression analysis
F factor
t factor
p-Value
RTD + CTD
5.146
0.029
0.043
Significant
RAA + CAA
0.409
0.879
0.398
Non Significant
_____________________________________________________________________________________________________________
*CTD = Control total dry mass
*CAA = Control actual ash value
*(Group 1-28); see appendix 3 for group key
Table II. Statistical summary of analysis of variance (ANOVA) among test species (Lymneae, Asellus and Gammarus)
______________________________________________________________________________________________________________
Epiphyton experiment data analysis
______________________________________________________________________________________________________________
Parameter of
Measurement
Population type
ANOVA pair
analysed
Mean
Standard
deviation
F Factor
p-Value
Inference
______________________________________________________________________________________________________________
Actual food
Consumption
(g)
High density
• Lymneae
1.4786
0.36037
16.108
0.000
Significant
•Asellus
0.7826
0.15335
• Gammarus
0.7410
0.08064
_______________________________________________________________________________________________________________________
Low density
• Lymneae
•Asellus
• Gammarus
1.7400
0.5210
0.5000
0.42816
0.21843
0.10019
31.360
0.000
Significant
______________________________________________________________________________________________________________
Food Consumption
at varying
densities
(g)
High density
(5 individuals
per replicate)
• Lymneae
• Asellus
• Gammarus
0.3550
0.0280
0.0322
0.10949
0.00300
0.00572
43.880
0.000
Significant
____________________________________________________________________________________________________________________-___
Low density
• Lymneae
• Asellus
• Gammarus
0.07455
0.00567
0.00498
0.03334
0.00254
0.00223
95.564
0.000
Significant
_______________________________________________________________________________________________________________
Weight-specific
Consumption
(g)
High density
• Lymneae
1.5782
0.39203
15.607
0.000
Significant
• Asellus
5.5758
1.77543
• Gammarus
4.3116
0.84069
_________________________________________________________________________________________________________________________Low density
• Lymneae
• Asellus
• Gammarus
2.2824
5.0806
4.7946
0.87795
2.24404
1.46619
4.469
0.000
Significant
________________________________________________________________________________________________________________
Table II. Statistical summary of analysis of variance (ANOVA) among test species (Lymneae, Asellus and Gammarus)......continues........
____________________________________________________________________________________________________________________
Dead leave experiment data analysis
____________________________________________________________________________________________________________________
Parameter of
Measurement
Population type
ANOVA pair
analysed
Mean
Standard
deviation
F Factor
p-Value
Inference
____________________________________________________________________________________________________________________
Consumption
Rate
(g)
Single species
treatment
• Lymneae
0.0279
0.01931
10.446
0.000
Significant
• Asellus
0.0095
0.00843
• Gammarus
0.0211
0.00721
_______________________________________________________________________________________________________________________________
• Lymneae + Asellus
0.0115
0.00530
1.045
0.358
Non Significant
• Lymneae + Gammarus
0.0100
0.00408
• Asellus + Gammarus
0.0124
0.00662
________________________________________________________________________________________________________________________________
Two species
treatment
Two & Three
Species treatment
• Lymneae + Asellus
• Lymneae + Gammarus
• Asellus + Gammarus
• Lymneae + Asellus +
Gammarus
0.0115
0.0100
0.0124
0.00530
0.00408
0.00662
0.0134
0.00703
1.232
0.304
Non Significant
Mesograzers dead leaf feeding experiment
Based also on three different parameters of measurement (Consumption rate; Replicate
total dry mass; and Replicate actual ash value), a total of 4 analysis (2 paired t-test; 1
regression analysis; 1 one-way ANOVA) conducted on meso-grazer test species revealed
significant differences (p < 0.05) in the amount of dead leaf disc consumed among the three
test species (Tables 1&2).
In 4, out of the total of 8 analysis (1 paired t-test; 1 regression analysis; 2 one-way ANOVA),
none significant differences occured in the same statistical tests mentioned above, while
taking into account, the same three parameters of measurement (Tables 1&2).
Fig 4. Effect of feeding on dead leaf disc during a 3-day feeding trial. Consumption rate; [Single species treatment; n=20 per
species]; [1 – 3 on x-axis], (1) Lymneae stagnalis ;( 2) Asellus aquaticus ;( 3) Gammarus pulex.
Consumption rate (CR); paired t-test
Measurement of the consumption rates for different pairs of grazer species indicated that
the feeding ability of Asellus aquaticus on alternative food source in the form of dead leaves
is highly significant. In table 1, the pairs of Lymneae and Asellus; Asellus and Gammarus
showed a highly significant feeding impact on dead leaf disc exposed to each test species
during the 3-day feeding trials at p < 0.000 respectively. There was no significant difference
in the feeding abilities of the pair of Lymneae and Gammarus on dead leaf disc at p < 0.144.
But at individual species feeding comparison, Lymneae stagnalis showed a highly significant
individual feeding ability over Asellus aquaticus and Gammarus pulex as indicated in figure 4.
This feeding character of Lymneae is no coincidence as it is confirmed in the paired t-test
results where it paired with Asellus and produced also a highly significant feeding effect on
dead leaf disc at p < 0.000 (Table 1).
Fig 5. Effect of feeding on dead leaf disc during a 3-day feeding trial. Consumption rate; [Two species treatment; n=20 per
species]; [1 – 3 on x-axis], (1) Lymneae stagnalis + Asellus aquaticus ;( 2) Lymneae stagnalis + Gammarus pulex; (3) Asellus
aquaticus + Gammarus pulex.
Fig 6. Effect of feeding on dead leaf disc during a 3-day feeding trial. Consumption rate; [Two & Three species treatment; n=20
per species]; [1 – 4 on x-axis], (1) Lymneae stagnalis + Asellus aquaticus ;( 2) Lymneae stagnalis + Gammarus pulex; (3) Asellus
aquaticus + Gammarus pulex; (4) Lymneae stagnalis + Asellus aquaticus + Gammarus pulex.
ANOVA test for dead leaf experiment
Here, a one-way ANOVA test detected a highly significant difference (p < 0.000) in
consumption rate for the single species treatment. However, consumption rate for both the
two and three species treatments were not significant as they showed values of none
significance at p > 0.358 and p > 0.304 respectively (Table 2). The none-significant feeding
effect of grazer species tested in combined treatments on dead leaf discs were also visually
possible to see in figures 5&6 where the Median values were almost on the same level in the
box plots.
Replicate total dry mas (RTD); regression analysis.
Measurement of replicate total dry mass (RTD) for the exposed dead leaf discs (groups 1-28)
indicated a significant difference (p < 0.043) over its control total dry mass (CTD) of
unexposed dead leaf discs counterpart (see appendix II for details) (Table 1). A graphical
confirmation of this significant difference is illustrated in figure 7.
Fig 7. Relationship between replicate total dry mass (RTD) and control total dry mass (CTD) of leaf disc after exposure to test
species during a 3-day feeding trial.
Replicate actual as value (RAA); regression analysis.
Measurement of replicate actual ash value (RAA) for exposed dead leaf discs (group 1-28)
indicated that there was no significant difference (p > 0.398) over its control actual ash value
(CAA) of unexposed dead leaf discs counterpart (see appendix II for details) (Table 1). A
graphical confirmation of this none significant difference is shown in fig. 8.
Fig 8. Relationship between replicate actual ash value (RAA) and control actual ash value (CAA) of leaf disc after exposure to
test species during a 3-day feeding trial.
4. Discussion (Not yet finished)
Analysis of data obtained from experiments in the course of this study indicated that
Lymneae stagnalis; Asellus aquaticus; and Gammarus pulex answered most parts of the two
hypothesis stated at the beginning of the study in the following ways;

The first hypothesis states “Mesograzer species under controlled laboratory
conditions, will consume epiphyton biomass at the same grazing rates”. Here, null
hypothesis was rejected following results obtained from single species treatment
analysis because most of the results indicated highly significant differences among
the species (Table 1&2).

The second hypothesis states “Mesograzer species feeding rates on epiphyton
biomass is proportional to the rate of feeding on dead leaves”. Here, null hypothesis
was accepted following results obtained from combined species treatment analysis
because most of the results indicated none significant differences among the species
(Tables 1 & 2).
Actual food consumption Vs Consumption rate.
These are two parameters used to measure the rates at which Lymneae, Asellus and
Gammarus grazed on algal biomass and the rate at which the same species fed on dead
leaves as alternative food source. Analysing data collected for the two parameters, results
from the ANOVA test for both parameters indicated a highly significant differences
(P < 0.000) exhibited among test species at the single species treatment level (Table 2). This
result treated and disproved the first hypothesis. It suggested that the grazing potential of
each test species used in the experiments is different from the others. The ANOVA test
(Table 2) confirmed this claim by revealing a highly significant difference among the species
at both high and low density populations. This was a typical example of the difference in the
grazing effects of each test species when exposed to the same quantity of algal (epiphyton)
biomass. Figures 1 – 3 shows a visual assessment of how the data collected for each test
species look like. In these figures, the Median values for each species vary clearly from the
other, hence further confirming the ANOVA test results which revealed highly significant
differences among the species.
The significant differences seen in the grazing impacts of these species on epiphyton as
confirmed by results obtained from this study are consistent with previous studies on similar
subject. Using a hypothetical example of the food preferences of mesograzers to a variety of
algal species to explain the high level of significant differences exhibited by the different
species used in this study, many studies have reported preference of certain algal species by
mesograzers over the others. For instance, Cruz-Rivera and Paul (2006) reported the
preference of mesogarzer species of marine origin (Cephalaspidean bubble snail) feeding
exclusively on cynobacteria.
Further more, the same ANOVA test results obtained from the dead leave experiment
revealed the same level of significant differences between test species feeding impacts on
dead leaf discs at the single species treatment analysis (Table 2).
Similarly, the second hypothesis was strongly supported by data obtained from this study.
Consumption rates at the combined species treatments of two and three species showed no
significant differences in the feeding impacts of mesograzer test species on dead leaf discs
(Table 2). More so, actual food consumption measured in the epiphyton experiment also
revealed two combinations of Asellus and Gammarus showing no significant differences in
the degree of grazing impacts both species exhibits on epiphyton biomass (Table I). Both
analyses suggest that there exist a significant difference in the rates that test species handle
different food sources. For example, the rate at which they graze on epiphyton is
significantly different to the rate at which they will feed on dead leaves.
One good thing about data on the second hypothesis is that it confirmed the abilities of
mesograzers to feed on a variety of food sources. The data is supportive of the notion held
by several authors who reported that many aquatic invertebrates are opportunistic and
generalists in their feeding habits as they tend to feed on other food sources within their
reach (Friberg and Jacobsen, 1994; Dangles, 2002). There is no doubt however, that similar
foraging behaviour can be exhibited by Lymneae stagnalis; Asellus aquaticus and Gammarus
pulex as was confirmed by this study.
Conclusion
The functional roles of invertebrate species in submerged vegetation can not be overemphasized. One important aspect of their functions in submerged vegetation ecosystem is
their ability to graze on epiphyton biomass thereby helping to maintain the ecosystem
balance in both physical and chemical properties that drive the ecosystem. This fact was
strongly supported by data obtained from this study.
References (Not yet ready).
Appendix 1: Dead leaves feeding trials illutration.
Experimental Set I
Single Species and Replicates (each species tested seperately): (i.e. Lymnaea, Gammarus,
Asellus)
Single sp
Control
Lymnaea
Single sp
Control 1
Asellus 1
1
1
2
2
2
2
Single sp
Control
Gammarus
3
3
3
3
4
4
4
4
Replicates
6 7 8 9 10
6 7 8 9 10
5
5
5
5
6
6
7
7
5
5
6
6
Replicates
8 9 10
8 9 10
11
11
11
11
12
12
12
12
13
13
13
13
14
14
14
14
15
15
15
15
16
16
16
16
17
17
17
17
18
18
18
18
19
19
19
19
20
20
20
20
Replicates
1
1
2
2
3
3
4
4
7
7
8
8
9
9
10
10
11
11
12
12
13
13
14
14
15
15
16
16
17
17
18
18
19
19
20
20
Experimental Set II
Combined Species and Replicates (two combination):
Lymnaea + Asellus
Combined sp exp.
Replicates
Control
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Lymnaea + 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Asellus
Lymnaea + Gammarus
Combined sp exp.
Replicates
Control
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Lymnaea
+ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Gammarus
Asellus + Gammarus
Combined sp exp.
Replicates
Control
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Asellus
+ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Gammarus
Experimental Set III
Combined Species and Replicates (three combination):
Combined sp exp.
Replicates
Control
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Lymnaea
+ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Asellus
+
Gammarus