Greater Parasitic Gregarine load in aquatic beetle larvae from polluted streams than pristine streams
By Sarah Di Bartolomeo
Human population growth and consumption have resulted in widespread pollution of tropical streams, yet, there are few studies examining the effects of pollution on aquatic invertebrates, which are often important bioindicators. Pollution severely impairs immune system function in a variety of organisms making them more susceptible to parasite colonization. Here, I examine the differences in colonization of Elmidae larvae by gregarine parasites in polluted and pristine stream environments. The stream habitat study sites were located in lower montane wet forest of Monteverde, Costa Rica. Twenty larvae were collected from two sites, both along the Quebrada Maquina. After larvae were collected, body length was measured and the peritrophic membrane of the gut was removed. Gregarine parasite abundance was determined by counting individuals after midgut staining. As expected by my hypothesis, individuals in the polluted environment had greater parasite abundance, greater numbers of parasites per cm of body length and were significantly smaller as larvae. This demonstrates that pollution is impacting gregarine parasite abundance in Elmidae beetles, specifically making hosts more susceptible to parasite infection.
Human domination in the Anthropocene has had profound effects on Earth’s ecosystems and biodiversity . This has resulted in extinction rates 100 to 1000 times greater than pre-human levels . More specifically, unrestrained human growth and consumption have resulted in increased human impact on freshwater systems for irrigation, hydroelectric power, and flood control . These anthropological impacts have had unparalleled effects on tropical stream ecosystems causing declines in biodiversity  and disruption of important ecological process like nutrient cycling, primary production, and decomposition .
Streams in highly populated areas that are subject to surfactant chemicals and wastewater dumping have higher concentrations of inorganic compounds and lower oxygen concentration, which places a large stress on stream communities . Pollution often results in cover of the substrata, sediment on the bottom of a stream, reducing available space for stream organisms . This pollution causes changes in biotic communities and species interactions, altering fragile stream ecosystems ; however, the full extent to which ecosystem structure is affected by anthropogenic disturbances remains largely unknown .
In many ecosystems, including streams, perturbations in structure and function (e.g. pollution) impact parasite transmission and abundance . Polluted environments severely impair immune system function in a variety of host organisms . Protozoan parasites, including gregarines, take advantage of the hosts’ compromised immune system and increase in colonization when under ecosystem stress . Typically, parasite species are most abundant at moderate pollution levels suggesting that they may serve as good indicators for earlier detection of unwanted environmental effects .
Because of this, parasite populations may serve as powerful bioindicators of environmental stress . Parasites can affect host fitness behaviorally, physiologically, or morphologically . This has been observed in a variety of species throughout the animal kingdom. Previous studies examining parasite colonization of chub communities in various levels of polluted environments found greatest parasite richness in polluted rivers when comparing to pristine habitats and environments with signs of pollution . Pollution also impacts parasite abundance of smaller organisms that are vital to stream ecosystems. Gut fungal parasite abundance of lotic black fly larvae was found to be in greater abundance in individuals from polluted streams than pristine .
Gregarine parasites (phylum Apicomplexa, class Conoidasida, subclass Gregarinasina) are large single-cellular parasitic protozoa commonly found inhabiting aquatic insect hosts . Hosts typically ingest gregarine oocysts containing infective sporozoites  which then attach and penetrate the hosts’ intestinal cells or reproductive system . Adults and larvae of family Elmidae (riffle beetles) are known hosts of gregarine parasites . For the purposes of this study, it was assumed that gregarine parasites affect hosts similarly to other parasites. Elmidae is also particularly sensitive to the degradation of streams and found at greater abundances in pristine habitats . This ability to react to stream quality reflects their ability as bioindicators due to a chance in parasite abundance depending on stream quality. Bioindicators are species that reflect the abiotic or biotic state of environment as well as serve to represent the impact of environmental change of an ecosystem . Elmidae larvae, found most often in well-aerated streams , contribute greatly to stream ecosystems by feeding on wood and creating highly grooved and sculptured surfaces . These spatially complex surfaces support diverse invertebrate communities .
To the best of my knowledge, there are no previous studies examining Elmidae and their response to anthropogenic impacts in the tropics. The purpose of this study is to determine whether Elmidae larvae have the potential to serve as bioindicators indicative of stream pollution. Here, I examine the difference in colonization of Elmidae larvae by gregarine parasites in polluted and pristine tropical stream environments. Based on previous studies, I hypothesize that individuals from polluted environments will have greater gregarine parasitic load, greater density of gregarine parasites as well as shorter body length, than individuals of pristine stream environments.
Elmidae larvae were collected from two sites in Lower Montane Wet Forest in Monteverde, Puntarenas, Costa Rica (Holdridge life zone). The first collection site (pristine) was an undisturbed stream habitat along the Quebrada Maquina at the Monteverde Biological Station at 1535 m in elevation (Fig. 1). The second collection site (polluted) was further downstream of the Quebrada Maquina, at an elevation of 1450 m, which experiences dumping of organics and other waste from residential areas (Fig. 1). Sites were chosen as there was access off lightly used trail.
Figure 1: Maps of polluted and pristine collection sites. (a). Overhead map of collection sites. The pristine sampling site is denoted by I and the polluted sampling site is II. (b). Topographical map, pristine sampling site denoted by I and polluted sampling by II.
Larvae from the pristine habitat were collected every 3 days beginning April 16, 2016 and concluding April 30, 2016 for a total of 5 days. Larvae from the polluted habitat were collected every 3 days beginning April 26, 2016 and concluding May 3, 2016 for a total of 4 days. Sampling was performed until 20 successful individuals were dissected from each site. All samples were obtained along a 20 m stretch of stream. At each site, a dip net was placed vertically into the substrate at the bottom of the stream. Rocks and sand were disturbed in front of the net and were collected into the net. Contents of the net were placed onto a white tray for examination and identification of Elmidae. Using tweezers, larvae were placed into jars containing 80 percent alcohol. Larvae were all dissected within 72 hours of capture and, if necessary, were refrigerated overnight to preserve the peritrophic membrane, a semi-permeable structure that forms the midgut of the larvae.
DISSECTION AND MICROSCOPY
Larvae were measured length-wise to the nearest hundredth of a centimeter under an Olympus CX22 dissecting microscope using a centimeter ruler. Body length was measured to approximate the length of gut. After measurement, a larva was placed in a Petri dish with a small pool of water containing a few drops of 1:4 dilution of Giemsa stain and buffer. Giemsa stain is commonly used to stain blood and bone marrow cells, as well as protozoan blood parasites. Using dissecting scissors, the ninth segment of the abdomen was removed to free the inferior end of peritrophic membrane of the midgut (Fig. 2a). The head was then removed from the prothorax using a pin. This served to separate the superior end of the midgut from the pharyngeal muscles. Using tweezers, the eighth segment of the abdomen was then squeezed to push the midgut out of the inferior end (Fig. 2b). Once a sufficient portion of the peritrophic membrane was visible, the midgut was removed by carefully grasping the end and pulling away from the abdomen. Following complete removal of the midgut, tweezers were squeezed along the membrane to completely clear the gut of feces content. This was also accomplished by gripping one end of the membrane with tweezers and repeatedly lifting out of a small pool of distilled water. The membrane was then colored with 1:4 dilution of Giemsa stain (Fig. 2c). After coloring, each larvae membrane was wet mounted and examined with an Olympus CX22 compound microscope at 400x magnification. Total number of gregarine individuals
were counted and recorded for each larva midgut (Fig. 2d).
Figure 2: Dissection process for the removal of the peritrophic membrane of Elmidae larvae. (a). General external anatomy of Elmidae larvae with labeled abdominal segments. (b) Removal of midgut from inferior end of larvae abdomen. (c) Peritrophic membrane stained with Giemsa after feces content removal. (d) Gregarine parasite found in peritrophic membrane of Elmidae larvae viewed at 400x magnification.
Statistic values are reported with mean and standard error. Unpaired Student’s t-tests were performed to analyze the comparison of individuals from the two sites. Analysis of covariance (ANCOVA) was conducted to determine if there was a significant difference between the number of gregarine parasites based on body length when controlling for habitat. All tests were performed in R and P<0.05 was regarded as significant.
Twenty larvae were collected from each habitat: pristine and polluted. Individuals from the pristine habitat ranged in body length from 1.1 – 1.4 cm and hosted between 0 – 7 gregarine parasites. Individuals from the polluted habitat ranged in body length from 0.7 – 1.25 cm and hosted between 1 – 7 gregarines. To the best of my knowledge, all gregarine parasites belonged to the same species as they were morphologically similar and found inhabiting Elmidae midguts.
The mean number of gregarine parasites counted on the peritrophic membrane of Elmidae larvae from polluted stream habitat (3.8 ± 0.4 SE individuals) was, on average, 33 percent higher than individuals collected from pristine habitat (2.55 ± 0.36 SE individuals; independent t-test, p = 0.026, t = 2.32, df = 37.6, n = 20 larvae/site; Fig. 3).
Figure 3: Gregarine parasites in the midgut of Elmidae hosts in pristine and polluted stream environments of Lower Montane Wet Forest of Monteverde, Puntarenas, Costa Rica. Polluted stream larvae possessed greater gregarine abundance than those of pristine stream habitat. Means are based on 20 individuals from each habitat. Error bars represent ± standard error.
The mean number of gregarines counted on the peritrophic membrane of Elmidae larvae, adjusted by gut length, from polluted environments (3.57 ± 0.36 SE individuals/cm) were, on average, 45 percent higher than those from pristine habitat (1.97 ± 0.4 SE individuals/cm; independent t-test, p = 0.003, t = 3.18, df = 35.6, n = 20 larvae/site; Fig. 4).
Figure 4. Gregarine number in the midgut of Elmidae hosts in pristine and polluted stream habitats of Lower Montane Wet Forest of Monteverde, Puntarenas, Costa Rica per centimeter of body length. Polluted stream larvae hosted greater abundance of gregarine per centimeter of body length than those of pristine stream habitat. Means are based on 20 individuals from each habitat. Error bars represent ± 1 standard error.
Elmidae larvae displayed a significant difference in body length between pristine and polluted habitats (independent t-test, p < 0.001, t = 5.3265, df = 32.769, n = 20 larvae/site). Individuals collected from pristine habitats (1.28 ± 0.02 SE cm) were, on average, 15 percent longer than individuals of polluted environments (1.1 ± 0.03 SE cm; Fig. 5).
Figure 5: Body length of Elmidae individuals in pristine and polluted streams of Lower Montane Wet Forest of Monteverde, Puntarenas, Costa Rica. Body length in centimeters was greater for individuals of pristine stream habitat than those from polluted areas. Means are based on 20 individuals from each habitat. Error bars represent ± 1 standard error.
An ANCOVA was conducted to determine if there was a significant difference between the number of gregarine parasites based on body length when controlling for habitat. It was determined that the number of gregarine parasites did not increase in proportion with increasing body length for either habitat (ANCOVA, F = 0.20, df = 1, p = 0.20, n = 20 larvae/site, Fig. 6).
Figure 6: Effect of body length on gregarine count in the gut of Elmidae larvae of pristine and polluted stream sites in Lower Montane Wet Forest of Monteverde, Puntarenas, Costa Rica. Gregarine number did not increase with body length for the pristine or polluted stream habitats. Pristine and polluted samples are each represented by 20 individuals. Each point represents one individual sampled.
This study demonstrated, consistent with my hypothesis, that gregarine parasites are more abundant in Elmidae hosts from polluted stream environments than those from pristine environments as found in previous studies with different host organisms. It was also demonstrated that gregarine infestation per centimeter of body length increased significantly in more polluted habitats when compared to pristine inhabiting individuals. Unsurprisingly, also consistent with my hypothesis, larvae from the polluted habitat were significantly shorter than those from pristine habitat.
These results clearly demonstrate that stream pollution is correlated with increased level of gregarine infestation of Elmidae midguts. Previous studies evaluating intestinal parasites of fish found that toxin exposure increases parasite burden . It is often found that parasites, like gregarines, with direct life cycles increase in abundance in contaminated habitats . Parasites with direct life cycles infect their host without an intermediate host where they reproduce and complete their entire life cycle. This is supported by the fact that pollution compromises host immune systems increasing susceptibility to parasite loads in a variety of organisms . Another possible explanation is the “density-dependent prophylaxis hypothesis” which states that as host densities increase and are more exposed to infective stages of parasites because of larger population sizes, hosts allocate more energy towards parasite resistance, making them less susceptible . Studies on Oriental armyworm moth (Mythimna separata) larvae found that virus-induced mortality declined from 95 percent for insects reared solitarily to 37 percent for insects reared at the highest density . This idea could potentially be applied to parasite transmission. Thus, since Elmidae abundances are greater in pristine habitats, due to Elmidae’s sensitivity to stream degradation, they will have greater resistance to parasite colonization in pristine habitats.
This study also shows that Elmidae larvae body length varied significantly between habitats with smaller individuals found inhabiting polluted stream habitats. While studies on Elmidae life cycles are somewhat lacking, it has been proposed that the larval cycle lasts one year with them hatching early summer which serves as a control for age . Therefore, smaller individuals with higher gregarine loads were found in polluted environments. Gregarine infected individuals experience delayed development and decreased longevity, especially under environmentally stressful conditions . Previous studies on earthworms (Lumbricus terrestris) determined that there was a significant negative relationship between parasite load and growth. Thus, parasite-mediated lower growth rate represents a fitness cost because mating success and choice is often correlated with size . This connective nature between pollution, increased parasites and inhibition of growth demonstrates the potential overarching impacts on the ecological function of Elmidae.
While larvae were identified by family level characteristics, there was no reason to believe that genus or species level differences would result in different outcomes due to strong morphological similarities and similarities in substrate between individuals collected from both sites. Therefore, there was no reason to believe the larvae were of different species despite my inability to classify them to species level.
In conclusion, elevated pollution levels are correlated with higher gregarine abundances in Elmidae hosts. This indicates that parasitism levels of Elmidae may serve as powerful bioindicators for stream water quality. Future studies sampling Elmidae from a range of stream qualities and determining if there is a correlation with parasitic load would shed light on Elmidae as an effective bioindicator. Furthermore, the ecological impact of parasitized Elmidae larvae would help complete the overall ecological picture of anthropogenic impact. In order to determine, While the overall ecological impact of higher levels of parasitism in Elmidae is still yet well understood, this study demonstrates, consistent with my hypothesis, that pollution is impacting gregarine parasite abundance in Elmidae beetles, specifically making hosts more susceptible to parasite infection.
I would like to thank my advisor Dr. Johel Chaves for his continuous guidance throughout the research process. Thank you to Dr. Alan Masters for helping develop my project idea and Moncho Calderón for his continuous patience and support in developing my methods and general encouragement. I would also like to thank Madison Cox for her advice and answering any experimental design questions. Finally, thank you to all CIEE staff and the Monteverde Biological Station for providing me with a field study site and facilities.
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