Effects of Microcystis aeruginosa on New Jersey Aquatic Benthic Macroinvertebrates

1 Thesis Signature 2 Title Page 3 Copyright Page 4 Acknowledgements 5 Table of


Effects of
25 Figure 4-The comparative mean survival percentage of mayflies, damselflies, and midges exposed and not exposed to Microcystis aeruginosa over a 36 day period, in intervals of every 6 days 26 Figure 5-The number of emerged mayflies, damselflies, and midges exposed to Microcystis aeruginosa compared to the number of emerged mayflies, damselflies, and midges not exposed to Microcystis aeruginosa 27 Figure 6-The mean survival percentage of mayflies exposed to Microcystis aeruginosa compared to mayflies not exposed to Microcystis aeruginosa over a 36 day period, in intervals of every 6 days 28 Figure 7-The mean survival percentage of damselflies exposed to Microcystis aeruginosa compared to damselflies not exposed to Microcystis aeruginosa over a 36 day period, in intervals of every 6 days 29 Figure 8-The mean survival percentage of midges exposed to Microcystis aeruginosa compared to midges not exposed to Microcystis aeruginosa over a 36 day period, in intervals of every 6 days 30

Introduction
Water Quality and Cyanobacteria The quality of freshwater around the world has declined in recent decades, decreasing the suitability for human use and for supporting healthy ecosystems. This poses a problem due to fresh water being an invaluable natural resource. Humans rely on freshwater every single day, using it to drink, bathe, and cook as well as for recreational and household purposes. Unhygienic and poor quality of water make up 3.1% of the world's deaths (Pawari and Gawande, 2015).
Fresh water also sustains wildlife, including those that are of ecological importance. Aquatic wildlife, particularly plants, insects, and fish, suffer from poor water quality as they can be sensitive to short-term changes. An excess of sediments, road salts, nutrients, and other contaminants can affect the ability for wildlife to persist in freshwater aquatic ecosystems, eventually altering the structure and dynamics of these delicate environments. One major factor related to the decline in water quality is an increase in cyanobacterial abundance in many freshwater systems (Sivonen and Jones, 1999).
Cyanobacteria are organisms with characteristics of both bacteria and algae; they contain blue-green pigments and, unlike other bacteria, perform photosynthesis, giving them the name "blue-green algae" (World Health Organization, 2009 (Sivonen and Jones, 1999), rendering these species as a common threat to the welfare of freshwater ecosystems throughout the United States, and especially in New Jersey (NJDEP, 2020). Microcystis spp. can produce the toxins Microcystins, Anatoxin-a, β-N-methylamino-L-alanine (BMAA), and Lipopolysaccharide (LPS) (Abeysiriwardena et al., 2018). Microcystin exposure in humans can lead to adverse health effects ranging from a mild skin rash to serious illness; unexplained sickness and death have been observed in dogs and other domestic animals that have come in contact with bodies of water containing high concentrations of microcystins (NJDEP, 2020).

Cyanobacterial Harmful Algal Blooms
When conditions are right, cyanobacteria can experience rapid population growth, called a cyanobacterial harmful algal bloom (CyanoHAB). CyanoHAB are a worldwide phenomenon, commonly occurring in freshwater, estuaries, and coastal marine ecosystems (Bricker et al., 2008). Occurrences of CyanoHAB have become common in watersheds primarily due to increases in anthropogenic sources contributing to heightened inputs of limiting nutrients (Huisman et al., 2005). Increases in limiting nutrients, such as nitrogen and phosphorus, can lead to eutrophication, which is a major stressor in freshwater ecosystems caused by runoff containing agricultural fertilizers, urban pollution, and sewage discharge (Stankovic et al., 2020). This enrichment of nutrients in waterbodies provides cyanobacteria the resources to rapidly multiply and create a bloom (Paerl, 2018). Water temperature is also a major contributing factor to CyanoHAB. Blooms usually occur from spring to fall when water temperatures are warm but can last year round, especially in New Jersey. Warmer temperatures prevent water from mixing in waterbodies that tend to be stagnant or slow movingincluding lakes, reservoirs, and pondswhich is known as stratification, allowing cyanobacteria to grow faster and in thicker mats (Hudnell et al., 2010). Climate change has intensified CyanoHAB around the world through warming and increasing hydrologic variability (Paerl, 2018). Changes in rainfall patterns have led to increased precipitation frequency and magnitude, heightening nutrient runoffs into waterbodies (Kunkel et al., 1999).  (Mcgeorge, 2020). 73% of those CyanoHAB events were above 100,000 cells/ml, the highest cell count being 56,300,000 cells/ml at Rosedale Lake (Mcgeorge, 2020). CyanoHAB are problematic for humans, reducing access to clean and safe water for drinking and recreational purposes (Qin et al., 2010), reducing fisheries production potential (Lee and Jones, 1991), and negatively affecting human health through cyanotoxin accumulation in edible fish tissue (Chueng et al., 2013).

Benthic Macroinvertebrates
CyanoHAB have led to the mortality of fish, benthic organisms, and other wildlife that live in and near freshwater ecosystems through direct cyanobacteria toxin exposure and bioaccumulation of toxins in food webs (Akamagwuna and Odume, 2020). One group that is particularly vulnerable to cyanobacteria are aquatic benthic macroinvertebrates, which are a vital trophic link between primary producers, detritus, and consumers (Poirier and Cattaneo, 2010).
Due to their high taxonomic diversity, position in freshwater ecosystem food webs, and variety of feeding habits and habitat tolerances, benthic macroinvertebrates are functional ecosystem health indicators (Akamagwuna and Odume, 2020), commonly used as bioindicators to assess ecosystem health and determine if an ecosystem is experiencing anthropogenic ecological degradation (Jonsson et al., 2018).
Benthic macroinvertebrates regulate water quality, decomposition, and nutrient cycling in freshwater ecosystems (Wallace and Webster, 1996). They comprise different functional feeding groups due to variation in behavioral mechanisms of acquiring food. These groups include scrapers, shredders, gatherers, filterers, and predators, all of which provide a balance to the trophic dynamics of a benthic ecosystem by consuming a variety of foods such as algae, detritus, leaf litter, suspended organic particles, and other small organisms (Barbour et al., 1999). When an imbalance of this community structure occurs, the detrital food web and rate of decomposition can be disrupted, slowing down the recycling of nutrients and organic material.
The United States Environmental Protection Agency (USEPA) has categorized benthic macroinvertebrates into three groups based on their tolerance to pollution: intolerant, moderately intolerant, and tolerant. CyanoHAB can lead to mortality in benthic organisms through clogged respiratory structures, oxygen depletion, and toxicity (Kroger et al., 2006). Cyanobacteria, specifically Microcystis spp., has led to mortality in invertebrates such as Daphnia spp. (DeMott et al., 1991), brine shrimp, Artemia salina (Metcalf et al., 2002), and copepods (Reinikainen et al., 2002). Microcystis spp. has also been observed to kill the aquatic mosquito larvae, Aedes aegypti (Kiviranta et al., 1993). However, an understanding of how cyanobacteria can disrupt communities of benthic macroinvertebrates across pollution tolerance groups remains incomplete.
This study aims to investigate the effect of Microcystis aeruginosa on the survival of benthic macroinvertebrates, across three pollution tolerance groups, intolerant, moderately intolerant, and tolerant. For this study, I tested immature stadia of the pollution intolerant taxa Ephemeroptera, mayfly nymphs, pollution moderately intolerant taxa Zygoptera, damselfly nymphs, and pollution tolerant taxa Chironomidae, midge larvae (Voshell, 2002), in a lab setting.
Mayflies, damselflies, and midges share a natural habitat in New Jersey freshwater ecosystems and are each in a different macroinvertebrate pollution tolerance group. Mayflies are considered to be important in freshwater ecosystems and are commonly used as ecological indicators as they are sensitive to environmental changes (Menetrey et al., 2008). Mayflies are primarily collectorgatherer detritivores but can also be herbivores and carnivores (McShaffrey and McCafferty, 1991). They mainly scrape periphyton off substrate and consume algae and detritus (McShaffrey and McCafferty, 1990). Mayflies are incredibly important in aquatic food webs as they are the main food source for many fish species (Morse et al., 1997). Damselflies are instrumental to aquatic ecosystems. Their life histories change when their habitats switch from aquatic to terrestrial environments; they are predators in aquatic ecosystems and prey in terrestrial ecosystems (Butler and deMaynadier, 2008). Midges are considered to be the most abundant and diverse group of benthic macroinvertebrates (Milosevic et al., 2013). Midges are opportunistic omnivores, consuming decomposed plant and animal matters, diatoms, and algae (Peckarsky et al., 1990). Their different feeding behaviors, usually gathering, filter feeding, and sometimes predatory, contribute to the recycling of organic matter, making them an integral part of aquatic food webs (Stankovic et al., 2020).
CyanoHAB may affect benthic macroinvertebrate community structure by reducing populations of less pollution-tolerant macroinvertebrate species, thus disrupting the composition of benthic macroinvertebrate communities. Such disruptions may affect the structure and function of entire food webs. The objective of this study is to determine if by exposing benthic macroinvertebrates from different pollution tolerance groups to Microcystis aeruginosa, as compared to the control group that was not exposed to Microcystis aeruginosa, the benthic macroinvertebrates would sustain a significantly higher mortality which would roughly correspond to their respective pollution tolerance levels.

Study Organisms
Ephemeroptera nymphs, Zygoptera nymphs, and Chironomidae larvae were collected from a manmade lake, Lake Wapalanne, in Sandyston, New Jersey in November of 2019.
Located in a watershed within forest landscape and protected land, Lake Wapalanne is fed by Big Flat Brook, with controlled outflow by a weir.
Experiments were conducted on either of the two Ephemerellidae genera, E. ephemerella and E. eurylophella, as these organisms naturally inhabit the benthos of Lake Wapalanne and its tributaries. E. ephemerella and E. eurylophella are considered scrapers and collectors, feeding on available periphyton found on substrate (Pennak, 1978). The Chironomidae species Chironomus riparius larvae were used in the experiment. This species, which are collector-gatherers, feed mainly on detritus (Pery et al., 2009). The experiment examined a combination of the three Zygoptera genera, Calopterygidae calopteryx, Coenagrionidae enallagma, and Lestidae lestes, as these organisms naturally inhabit the benthos of Lake Wapalanne and its tributaries, and have similar habitat preferences, feeding behaviors, and life histories (Pennak, 1978). C. calopteryx, C. enallagma, and L. lestes are predators in their environment, feeding mainly on small invertebrates (Pennak, 1978). Benthic macroinvertebrates were randomly allocated into 30.5 centimeters tall plastic pipes, each with a diameter of 7.6 centimeters ( Figure 1). The three macroinvertebrate pollutiontolerance groups were replicated four times in each bin, making a total of twelve plastic pipes per bin; there were fifteen individuals in each plastic pipe. Two sections, each measuring 5 centimeters wide and 25.4 centimeters long, were cut out of plastic pipes and covered with fiberglass mesh (pore size 1mm²), to allow water and Microcystis aeruginosa cells to flow freely through plastic pipes but keep macroinvertebrates inside (Figure 1). Gravel and Potamogeton perfoliatus (claspingleaf pondweed) were taken from the lake and placed in the plastic pipes to mimic a natural lake environment.
CyanoHAB usually take place in the summer and fall months when water temperature is warm, therefore experiments were conducted in a temperature-controlled greenhouse.
Cyanobacteria and its growth rate show a strong response to high water temperatures, usually above 25 degrees Celsius (Paerl, 2018); thermometers and water heaters were used to maintain a water temperature of 20 to 25 degrees Celsius. Aerators were used to keep water oxygenated and circulated. In this study, the treatment group was inoculated with M. aeruginosa at a concentration of approximately 100,000 cells/ml. Microcystis aeruginosa cell count was monitored every three days and maintained as needed using a microscope. Macroinvertebrate survival was checked every six days for a 36-day period to determine macroinvertebrate survival and mortality. Individuals were confirmed dead by the absence of body or gill movement.
Macroinvertebrates that emerged into terrestrial adults during the experiment were considered alive during data analysis.

Statistical Analysis
To test for differences in survival percentages among the treatments (insect type and cyanobacteria presence), I used a generalized linear model with a binomial distribution which approximates a logistic regression with categorical data. A binomial distribution is often used when testing for differences in percentages or ratios among groups. I then conducted a Tukey's posthoc analysis to test for specific differences among the groups, comparing each species and the treatment and control groups for each species. One-way ANOVAs were used to test for differences in means in total surviving macroinvertebrates between control and treatment groups.
A p-value of less than 0.05 is considered statistically significant for this study. For this analysis, I used the glm and TukeyHSD functions, along with the Anova function in the car package in R.  (Figure 2). The Tukey's test also revealed a significant difference between midge survival percentages and the survival percentages of both mayflies and damselflies, but the difference between mayfly and damselfly survival rates was not statistically significant. This demonstrates that mayflies (pollution intolerant) and damselflies (moderately intolerant) had similar responses to Microcystis aeruginosa in terms of mortality. In the treatment, there was no significant difference between the survival percentages of each macroinvertebrate pollution tolerance group.

Benthic Macroinvertebrate Survival
The mean survival percentage for treatment groups exposed to Microcystis aeruginosa was substantially lower than the mean survival percentage for control groups not exposed to Microcystis aeruginosa (p<0.001). Mayflies had a survival percentage of 38% in the Microcystis aeruginosa treatment group compared to a 98% survival percentage in the control group ( Figure   3). Damselflies had a survival percentage of 43% in the Microcystis aeruginosa treatment group compared to a 98% survival percentage in the control group ( Figure 3). Midges had a survival percentage of 72% in the Microcystis aeruginosa treatment group compared to a 100% survival percentage in the control group (Figure 3).
In the treatment group, the percentage of individuals that died corresponded with their respective pollution tolerance levels: mayflies (pollution intolerant) died earliest on average and in the highest total number of deaths, and midges (pollution tolerant) died the latest on average and in the lowest total number of deaths, while these values for damselflies (moderately intolerant) fell in between (Figure 4). In the control group, there was only one death recorded for mayflies and damselflies, which occurred at the seventh week (Figure 4).

Number of Surviving Insects:
These results can also be demonstrated in terms of mean surviving insects of 15 macroinvertebrates, including insects that emerged (Table 2). A notable dissimilarity in the number of emerged macroinvertebrates occurred between treatment and control groups. Among treatment mayflies, zero emerged, whereas seven emerged in the control group. One damselfly emerged in the treatment group, and nine emerged in the control group. No midges emerged in either group. The several emerged individuals in the control group could be a result of normal macroinvertebrate growth in a healthy environment. Microcystis aeruginosa appeared to have a substantial effect on either the growth or ability to emerge for certain macroinvertebrates ( Figure   5). Due to the emergence of insects, some individuals did not remain present in the full experiment, which should be taken into consideration.

Discussion
A previous study on the survival of mayflies (Smith et al., 2007) when exposed to microcystin-LR documented similar findings, where 20% of egg hatchlings died in a seven-day period when exposed to lower toxin concentrations of 0.01 μg/ml; 100% of egg hatchlings died within five days when exposed to a higher concentration of microcystin-LR of 10.0 μg/ml.
Microcystis aeruginosa had a negative effect on the survival of the macroinvertebrates in this experiment, reducing the survival by more than 60% in the most sensitive group, Ephemeroptera.
Mayflies are particularly sensitive to poor water quality and pollution and had the lowest survival percentage, 38%.
In a previous study where the different mayfly larval stages were exposed to microcystins, the eggs required two extra days for 90% to emerge (Smith et al., 2007). In addition, 20% of the egg hatchlings (young nymphs) exposed to typical bloom conditions (0.01 μg/ml) died within a seven-day period at the highest concentration (10 μg/ml), 100% of egg hatchlings died after five days (Smith et al., 2007). Microcystins, therefore, had a negative effect on the emergence time of mayfly eggs and the survival of egg hatchlings thereafter. A similar response occurred in this study where Microcystis aeruginosa delayed or prohibited nymphs from emerging into terrestrial insects. Since benthic macroinvertebrates have different larval stages that are a part of aquatic and terrestrial ecosystems, exposure could affect both food webs in a natural setting.
CyanoHAB have been shown to reduce feeding rates for not only filter feeding taxa (Yang et al., 2006), but for visual predators due to reduced water quality and light (Jiang et al., 2014). Damselflies are visual predators and thrive in waters that have limited suspended particles, high visibility and high oxygen levels. CyanoHAB create an unfavorable environment for damselflies, with increasing suspended particles, decreasing visibility, and deoxygenating bottom waters (Akamagwuna and Odume, 2020). Midges and mosquito larvae are both pollution tolerant. In a study, where mosquito larvae were exposed to Microcystis aeruginosa and toxic strains of Anabaena circinalis and Oscillatoria agardhii, the larvae developed lesions in their epithelial cells just after a 24-hour exposure period (Saario et al., 1994). Microcystis aeruginosa and cyanotoxins that it produced can affect benthic macroinvertebrates by being ingested or diffused across the cuticle, egg, or gill membranes (Smith et al., 2007). However, the exact nature of the effects of Microcystis aeruginosa and other cyanobacteria exposure is difficult to determine due to the limited information available regarding the toxicity to benthic macroinvertebrates. A consideration for future research on the effects of Microcystis aeruginosa on benthic macroinvertebrates is that studies should take into account that each macroinvertebrate species likely has a unique tolerance to Microcystis aeruginosa. Although Microcystis aeruginosa had an effect on these specific species of macroinvertebrates, that may not be the case for other species of the same taxonomic order.  (Strayer, 2006) and ubiquitous (Voelz and Mcarthur, 2000) organisms in freshwater ecosystems, giving them a vital ecological role. Because benthic macroinvertebrates are an important trophic link between primary producers and consumers (Poirier and Cattaneo, 2010), a shift in abundance and diversity could affect each trophic level to which these macroinvertebrates are connected (Maisto et al., 2017).
As anthropogenic eutrophication and climate change increase the magnitude and frequency of CyanoHAB events around the world, the need to understand how blooms affect aquatic ecosystems is paramount (Briland et al., 2020 unique reaction to cyanobacteria. Although Microcystis aeruginosa had an effect on these specific species of macroinvertebrates, that may not be the case for other species of the same taxonomic order. Moreover, cyanobacteria seem to have the ability to take advantage of an increasingly warming world. Researchers should focus on how this advantage will affect freshwater ecosystems ecologically and economically in the future as bloom events continue to increase.   Figure 4-The comparative mean survival percentage of mayflies, damselflies, and midges exposed and not exposed to Microcystis aeruginosa over a 36 day period, in intervals of every 6 days.
Figure 5-The number of emerged mayflies, damselflies, and midges exposed to Microcystis aeruginosa compared to the number of emerged mayflies, damselflies, and midges not exposed to Microcystis aeruginosa. Figure 6-The mean survival percentage of mayflies exposed to Microcystis aeruginosa compared to mayflies not exposed to Microcystis aeruginosa over a 36 day period, in intervals of every 6 days. Figure 7-The mean survival percentage of damselflies exposed to Microcystis aeruginosa compared to damselflies not exposed to Microcystis aeruginosa over a 36 day period, in intervals of every 6 days. Figure 8 -The mean survival percentage of midges exposed to Microcystis aeruginosa compared to midges not exposed to Microcystis aeruginosa over a 36 day period, in intervals of every 6 days.  Table 2. Mean surviving mayflies, damselflies, and midges exposed to Microcystis aeruginosa compared to mean surviving mayflies, damselflies, and midges in the treatment group.