The River Rwizi catchment, located in South-western Uganda, is a crucial hydrological and ecological region facing various environmental challenges. Spanning approximately 8554.7 km², the catchment is situated between longitudes 30.21˚ E to 32.52˚ E and latitudes 0.24˚ S to 0.92˚ S, with an altitudinal range of 1300 to 2170 meters above sea level [36] [37] . The river, stretching about 55 km, meanders eastwards from the hilly terrain of the region, ultimately discharging into Lake Victoria.

The upper catchment of the River Rwizi traverses’ multiple districts, including Buhweju, Sheema, Bushenyi, Mbarara, Ntungamo, Isingiro, and Kiruhura, before it flows into Lake Mburo (Figure 1). This region experiences a bi-modal climate characterized by distinct wet seasons from March to May and September to November, providing essential rainfall for the river and the surrounding ecosystems [38] . The warm temperatures, ranging between 20˚C - 30˚C year-round, are attributable to the equatorial proximity of the area.

The site selection and delineation process occurred during district consultation workshops, relying on local land uses and identifying areas with potential pollution

manifestations. Active community participation was encouraged, leading members to propose sampling sites based on their local knowledge, considering accessibility and proximity to the main River Rwizi. The main river was segmented to capture the diversity of the riverine environment. Strategically, twelve sites were chosen for macroinvertebrate sampling, with two data collection points designated for each river segment to account for variations along the segment’s length (Figure 1). The selection aimed to balance representativeness across different segments, ensuring a comprehensive assessment of macroinvertebrate biodiversity within the River Rwizi watershed. This approach not only effectively involved the community in the research process but also nurtured a sense of ownership and collaboration, ultimately enhancing the scientific rigor of the study.

A mixed-methods approach for macroinvertebrate metrics and socio-ecological dynamics in the context of a citizen science BioBlitz research design that combined both quantitative and qualitative research methods was used in this study.

Permission to conduct the research was obtained from district authorities, facilitated by the District Chief Administrative Officers. The research team organized a one-day consultative workshop to introduce the project to security leaders, district political heads, and technocrats. This workshop served as a platform to disseminate the research concept and its objectives. The outcomes of these workshops were pivotal in publicizing the project to technocrats at the district level and establishing an entry point into the communities within the riparian zone of River Rwizi parishes. Subsequent introductions were made to extension workers, parish chiefs, and Local Councils.

The extension workers, parish chiefs, and Local Councils played crucial roles as focal points for community entry and engagement. They actively participated in the selection of participant volunteers for the Citizen-Science-BioBlitz activity. Furthermore, they guided the mobilization and recruitment processes of participants for data collection. Participation was restricted to individuals above 18 years old, and voluntary acceptance was sought from potential participants. Those who agreed underwent training in macro-invertebrate data collection methods. This training was particularly important in the context of deteriorating water quality, anthropogenic pollution, and the overall need for water conservation in the River Rwizi watershed.

To ensure the safety of participants, all necessary safety precautions were implemented, including strict adherence to the National Guidelines for Research involving Humans as Research Participants [39] .

A guided bio-blitz sampling strategy was implemented to conduct an inventory of macro-invertebrates along selected sites of the River Rwizi. The lead team of bio-blitzers was composed of biologists from Mbarara University of Science and Technology (MUST), including expert lecturers, three technicians, two graduate students, and two trainees from the International Crane Foundation (ICF). The involvement of expert academic staff from MUST ensured scientific rigor and proficiency in the sampling process. Additionally, community volunteers, comprising 6 to 8 individuals from each of the 12 selected sites along the River Rwizi, actively participated in the bio-blitz events. The engagement of community volunteers not only expands the data collection capacity but also aligns with the principles of citizen science, enhancing community involvement in scientific research [11] . The collaborative effort between academic experts and community volunteers aimed to generate comprehensive data on macro-invertebrates.

The training process commenced with an orientation for the Mbarara University of Science and Technology (MUST) team, providing them with a comprehensive understanding of the natural history of the River Rwizi, emphasizing the significance of aquatic biodiversity, and elucidating the socio-ecological role of macro-invertebrates in aquatic environments. This foundational knowledge is aimed at facilitating experiential learning, community science education, and research purposes. Subsequently, the team conducted a field reconnaissance as part of a pilot study to validate the most suitable sampling sites along the river. This involved a detailed description of the sites, incorporating input from community participants. During this phase, team members were introduced to the technical procedures involved in the collection and treatment of samples, as well as the utilization of protocols and taxonomic guides for the identification of aquatic macro-invertebrates. This preparation phase was crucial to orient the team and equip them with the necessary skills to subsequently train local community volunteers for participatory citizen Science-BioBlitz surveys. The overarching goal was to determine the macro-invertebrate assemblages for each identified river segment.

The sampling strategy implemented in this study encompassed nine sampling sessions at each designated site between January and July 2023, with a focus on macroinvertebrate organisms. Utilizing a modified D-frame metal sieve with a prescribed mesh size of 5 mm, the sampling adhered to well-established macrobenthos sampling protocols [40] . Various methods, including scoops, dips, or sweeps, were employed along the littoral river bank areas recognized as the most productive habitats for macro-invertebrates. To maintain data accuracy, organisms were meticulously rinsed by sweeping them against the flowing river water current, with any debris and non-macro-invertebrates, such as fish, released back into the water. The collected macro-invertebrates underwent further processing, involving hand-picking from the sieve using forceps or brushes. Subsequently, they were spread on a white sheet of absorbent paper for participatory taxonomic identification and counting. This detailed and systematic approach aimed to provide a representative understanding of the macroinvertebrate taxa present in the River Rwizi.

The macroinvertebrate sampling and identification procedures were conducted by adhering to standardized techniques to ensure accuracy and reliability. Citizen scientists and volunteers, equipped with prior training, skillfully carried out hands-on sampling using essential equipment like D-nets, trays, and containers for the systematic collection of macroinvertebrate specimens. Following the guidelines established by USEPA [41] , samples were methodically composited for each entire site and subsequently pooled for a comprehensive analysis spanning different river segments.

The taxonomic identification and verification processes were collaborative efforts involving university experts and students, guided by identification guides [42] . During the verification stage, the team actively engaged the community, fostering a culture of shared learning and collaboration. Unidentified taxa, meticulously preserved in 70 percent ethanol, were transported to the laboratory for further processing and rigorous identification. This systematic collection and identification procedure were replicated for additional samples randomly gathered at various locations during one-hour sampling sessions.

In the laboratory, the utilization of stereo microscopes and hand lenses facilitated the observation of finer details for taxonomic identification, specifically at the family taxon level. The specifications and justifications for family taxa were grounded in established studies and aligned with USEPA [41] protocols, as well as guidelines from Fry [42] . It is noteworthy that this comprehensive approach considered the inherent taxonomic challenges associated with the absence of published identification guides for aquatic macrobenthos in East Africa [43] . Collectively, this rigorous methodology ensured a thorough understanding of the macroinvertebrate taxa present in the River Rwizi.

Community participants were actively encouraged to provide feedback and reflections on their BioBlitz experiences. Surveys and interviews were conducted to capture qualitative data on community perceptions, satisfaction, and awareness. The study employed an open citizen science method, fostering natural discussion, to collect feedback from participants about their individual and collaborative BioBlitz experiences. The primary aim was to investigate shifts in awareness, knowledge, and attitudes towards aquatic biodiversity. Narratives capturing the emotional dimensions of BioBlitz events were documented. Community participants’ BioBlitz experiences were opportunistically gathered from their narratives during or after field sampling. The analysis categorized these experiences as positive (supportive of BioBlitz participation) or negative (critical or unsupportive).

Thematic content analysis was conducted to characterize the typologies of pollution concerns in selected riparian areas along specific river segments of the River Rwizi. This involved the integration of community observations and narratives, capturing various types and sources of river pollution and degradation as unanimously agreed upon by the community through group consensus.

Quantitative analysis involved computing the composition and abundance of invertebrate orders and families within specific river segments and sample sites. Paleontological Statistics Software (PAST) was employed for this purpose

The Shannon-Wiener Diversity Index (H’) was used to measure taxonomic diversity by considering both the variety of taxa and their even distribution. The Dominance Diversity Index (D) was also calculated to assess the abundance of dominant taxa in a community. Additionally, pair-wise Sorensen indices of similarity (S) were determined to explore the connections between taxa at different sites within river segments or across multiple sites. The Sorensen Coefficient (SC) calculator [44] was employed to compute similarity quotients based on species presence, enabling the comparison of species composition between different communities.

The study reveals notable variations in riparian characteristics along the River

Rwizi, primarily driven by differential degradation and pollution. These variations can significantly impact macro-invertebrates, including aquatic insects, crustaceans, and mollusks, residing in freshwater ecosystems. Nseka et al. [50] found that land use and cover characteristics in the upper Rwizi macro-watershed exhibited spatial and temporal variations affecting hill slope hydrological characteristics in the watershed due to increased human activities with consequent severe degradation in the area [50] .

The study found high insect diversity and abundance in the River Rwizi, with Odonata, Hemiptera, and Ephemeroptera as the dominant orders. These insects are sensitive to water quality and play important roles in freshwater ecosystems [4] [51] [52] [53] . However, the absence of Plecoptera and the scarcity of Trichoptera suggest that the river may be polluted by human activities [46] [47] [48] [49] . The EPT index may not capture the river’s health, and more water quality parameters are needed, a limitation perhaps posed by the BioBlitz approach.

This study systematically explored the macroinvertebrate biodiversity and socio-ecological dynamics in six distinct segments along the River Rwizi, including rural hilly upstream, rural upstream wetlands, rural flood plains, peri-urban upstream, urban sprawl, and peri-urban downstream regions. This investigation provided comprehensive insights into the diverse ecological conditions and anthropogenic influences representative of different parts of the river. Anthropogenic influences, including pollution, habitat alterations, climate change, species introductions, and physical modifications, emerge as substantial threats to freshwater ecosystems, exerting profound effects on macro-invertebrates through mechanisms such as alterations in abundance, health, survival, reproductive success, life cycles, distribution, biodiversity, migration challenges, and nutrient-induced eutrophication [54] .

The absence of a clear pattern in taxa metrics along the River Rwizi continuum, from the upstream to the urban sprawl and peri-urban downstream segments, indicates a complex interaction of ecological, environmental, and anthropogenic factors. The variability in macroinvertebrate richness, diversity, and evenness across different river segments challenges simplistic assumptions about water quality trends based on geographical locations. This suggests that the influences on macroinvertebrate communities are multifaceted, with ecological and anthropogenic factors potentially offsetting each other’s effects. Doretto et al.’s [55] findings emphasize the need for a nuanced understanding of the factors shaping freshwater ecosystems, considering the intricate interplay of natural and human-induced dynamics along the River Rwizi.

Previous research has highlighted the influence of both habitat complexity and heterogeneity on river macro-invertebrates [56] [57] . In this context, the rural upstream sections, such as RHU, may exhibit simpler and less varied habitats compared to downstream or more urbanized sections. The reduced habitat complexity in RHU could contribute to the observed lower diversity, as the literature suggests that a lack of habitat complexity tends to reduce overall diversity in river ecosystems.

The application of Sørensen’s index to assess taxonomic composition similarity among river segments offers valuable insights into the ecological relationships within the River Rwizi. The high dissimilarity between Rural Hilly Upstream (RHU) and Rural Flood Plains (RFP) suggests significant differences in the macroinvertebrate communities between these two segments. This dissimilarity might be attributed to variations in habitat types, water quality, or other ecological factors. On the other hand, the high similarity between Rural Flood Plains (RFP) and Peri-Urban Upstream (PUU) implies a closer taxonomic resemblance, indicating potentially shared ecological conditions or similar macroinvertebrate compositions in these river segments. These findings underscore the heterogeneity of ecological conditions along the River Rwizi, emphasizing the importance of considering specific river segments when implementing conservation and management strategies.

Community reflections and feedback on BioBlitz experiences, as analyzed thematically, unveil a spectrum of both positive and negative sentiments. On the positive side, BioBlitz events have demonstrated notable success in cultivating positive experiences, engaging participants effectively in ecological activities that offer hands-on experiences beyond traditional learning methods. The incorporation of educational components has played a pivotal role in this success, as participants value the interactive opportunities to learn about local ecosystems, biodiversity, and environmental conservation, fostering not only knowledge but also environmental awareness. Positive responses also indicate that BioBlitz events act as catalysts for community collaboration, nurturing a sense of shared engagement and collective effort in understanding local ecosystems, thus enhancing the social dimension of ecological awareness and emphasizing shared responsibility for environmental stewardship. However, amid overwhelmingly positive sentiments, some participants express concerns about environmental issues, emphasizing the need to translate awareness into concrete action and advocate for proactive conservation measures.

On the negative side, the study’s findings reveal a complex set of attitudes and perceptions among participants that influence their engagement with the educational and scientific aspects of the research. Participants prioritize survival needs over educational components, perceiving learning experiences as less significant and secondary to immediate concerns. Accusations of hypocrisy and deception toward researchers signify a substantial level of distrust, with suspicions of financial motives. Resistance to acknowledging water pollution and a firm assertion of water cleanliness suggest a potential disconnect between participants’ perceptions and environmental realities, contributing to apprehension and distrust within the community. Perceptions of an education gap highlight broader issues of accessibility in scientific engagement, with specific biases and aversions potentially limiting participants’ ecological awareness. Women participants citing time constraints and expressing a perceived lack of relevance or interest underscore gender-specific challenges affecting participation. Lastly, skepticism about researchers’ commitment and past observations of transience contribute to an overall anticipation of potential abandonment, emphasizing the need for trust and long-term relationships in successful research endeavors.

The citizen science BioBlitz approach emerges as a valuable tool for macroinvertebrate documentation, promoting community engagement and fostering awareness among local residents [29] . Involving citizens in the data collection process enhances understanding of local biodiversity [58] , and the large-scale participation facilitated by this approach allows for comprehensive spatial coverage and data collection over extended periods [17] . The democratization of science through community involvement [11] and the generation of valuable datasets for long-term monitoring and ecological research [28] further emphasize the significance of the citizen science BioBlitz approach in strengthening the connection between local communities and their ecosystems. Accordingly, the citizen-science-BioBlitz approach is a novel and promising method for monitoring river macro-invertebrates, which are important indicators of water quality and aquatic ecosystem health. By engaging volunteers to collect and identify macro-invertebrates at multiple sites and times, this approach can generate large-scale and synoptic data on the diversity and distribution of these organisms, as well as their responses to environmental changes and human impacts. This approach can also enhance the communication and collaboration between scientists, authorities, and communities, and foster public awareness and involvement in river conservation and management. However, this approach also faces some challenges and limitations, such as the quality and reliability of the data, the training and support of the participants, and the evaluation and feedback of the outcomes and impacts. Therefore, this approach requires careful planning, implementation, and improvement to ensure its effectiveness and sustainability.