Morphometric analysis of black fly larvae (Diptera: Simuliidae) in Tijuca National Park, Rio de Janeiro, Brazil: A potential tool for biomonitoring aquatic environments

Morphometric analysis of black fly larvae (Diptera: Simuliidae) in Tijuca National Park, Rio de Janeiro, Brazil: A potential tool for biomonitoring aquatic environments | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Morphometric analysis of black fly larvae (Diptera: Simuliidae) in Tijuca National Park, Rio de Janeiro, Brazil: A potential tool for biomonitoring aquatic environments Tainá Maria Miranda Souza Martins, Agatha Alves da Silva, Barbara Alves Victer, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8205131/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Simulids are dipteran insects that colonize freshwater environments in their immature life stages. This study aims to compare the body size of Simulid larvae of the species Simulium pertinax and Simulium (Inaequalium) sp. collected in waterways inside and outside of Tijuca National Park. Collections were made in 2019 and 2021. Abiotic variables were verified in situ using specific equipment, and a protocol was also applied to assess the integrity of the river waters in each river. A total of 1,311 larvae were collected and found to be the following species: S imulium pertinax, Simulium (Inaequalium) sp., Simulium (Psaroniocompsa) sp., Simulium (Psaroniocompsa) anamariae, and Simulium (Psilopelmia) perflavum . The average body, head, and protorax leg sizes of the larvae were larger in the outer section of the Tijuca River than in the inner section. The Wilcoxon test found a significant difference (p-value < 0.05) in the body size of S. pertinax and S. (Inaequalium) sp. compared to 2019 and 2021. The CCA also showed that the body, head, and propata sizes of S. pertinax were influenced by river brightness and altitude. The correlations between the abiotic variables and morphometric measurements were highly significant. As there are still few studies linking the morphometry of blackfly larvae to river water integrity, this work stands out for advancing in this field. Our results reinforce the potential of these organisms as biological indicators and point to new pathways for the biomonitoring of aquatic environments. Simulids Morphometric analysis Biomonitoring Environmental indicators Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction Water is an essential natural resource not only for humans, but for all living beings, harboring enormous biodiversity in aquatic ecosystems. Surface freshwater, found in rivers and lakes, represents only about 0.01% of the total water on Earth (Kuhlmann et al. 2019). However, this ecosystem can be impacted by anthropogenic actions that compromise the quality of these water bodies, such as deforestation of riparian forests, pollution from industry, households and agriculture, and the introduction of exotic species, among others (Baptista, et al. 2000 ). According to CONAMA Resolution 001 of 23 January 1986, an environmental impact can be defined as “any change in the physical, chemical and biological properties of the environment resulting from human activities that directly or indirectly affects [...] the quality of environmental resources”. With this, it is increasingly apparent that studying the environmental impacts caused by human activities on water bodies is vital for their management and administration, which can ensure more effective decision-making in their protection or recovery (Jacinto-Junior and Lucena 2022 ). To this end, constant biomonitoring is necessary to detect these environmental impacts in their early stages, since once serious water degradation has been reached, it becomes difficult to reverse the scenario (Baptista 2008 ). The assessment of biotic communities for the purpose of investigating ecosystem conservation through environmental monitoring and bioindicators has proven to be an alternative for detecting impacts resulting from human activity (Docile and Figueiró 2013 ; Silva and Viana 2021). Among the different bioindicator organisms, the order Diptera is considered a good environmental indicator because they are abundant in lotic or lentic aquatic environments and can tolerate high concentrations of organic matter in waters with low oxygenation (Lima et al. 2017 ). Included in this order are insects of the Simullidae family, also known as simulids. The larvae of these insects are filter feeders, and their diet consists of organic matter dissolved in the water column (Bernotiene 2006). Among the Diptera, simuliids, also known as black flies, are a dipteran family that breeds in lotic systems (Hamada and McCreadie 2002, Landeiro et al. 2009 , Figueiró et al. 2014 , Menzel et al. 2019 , Lima-De-Sousa et al. 2024 ), feeding on suspended organic matter in the water column. There are studies that mention simuliids as environmental bioindicators, meaning they can be used as a tool to assess the ecological status of watercourses in urbanized areas (Couceiro et al. 2014 , Ciadamidaro et al. 2016 ). This occurs because anthropized sites may favour certain species (Coppo and Lopes 2010; Docile et al. 2015 ; Costa et al. 2024 ). The literature reports that some species, such as Simulium pertinax Kollar, 1832, can develop in aquatic environments impacted by varying degrees of pollution (Strieder et al. 2006 ). One of the influences that an impacted environment can exert is on the body size of simulid larvae. Ciborowski and his collaborators (1997) observed that the availability of organic matter in water can influence a variation in the body size of simulid larvae, while Figueiró et al ( 2015 , 2021 ) and Pinto (2024) observed the phenotypic plasticity of these organisms among different microhabitats. This study considers the premise that water bodies located within Tijuca National Park will have a better conservation status than those located outside the park, since it is a fully protected conservation unit (ICMBIO et al. 2008). Collection sites were therefore selected both inside and outside this conservation unit. Tijuca National Park has several water sources that are used to supply the homes surrounding the park and are also used for recreational purposes (Silva et al. 2017 ). Within the park, there is also a water treatment plant called Afonso Viseu, which is responsible for part of the drinking water supply for Alto da Boa Vista (CEDAE 2018 ), highlighting the importance of conserving the water resources of Tijuca National Park and the fundamental role of environmental monitoring in ensuring the monitoring and protection of water bodies. Therefore, this study aims to evaluate how the conservation status of water bodies inside and outside Tijuca National Park influences the abundance and body size of Simuliidae larvae, contributing to the understanding of how environmental quality affects these bioindicator organisms. 2 Materials and Methods 2.1 Study area Tijuca National Park (PARNA-Tijuca), created in 1961 initially under the name Rio de Janeiro National Park (BRAZIL 1961), is a fully protected conservation area under Law No. 9,985 of July 18, 2000, covering an area of 3, 953 hectares located between parallels 22°55' South and 23°00' South and meridians 43°12' West and 43°19' West, in the south-central part of the state of Rio de Janeiro (ICMBIO 2008 ). According to Souza ( 2015 ), on a global scale, PARNA-Tijuca is one of the largest replanted forests located in an urban area. PARNA-Tijuca is divided into four main areas that can be differentiated according to their state of conservation, land use, and environmental characteristics. These sectors are called: Serra da Carioca, Floresta da Tijuca, Pedra Bonita/Pedra da Gávea, and Pretos-Forros/Covanca (Zago et al. 2020). The location of PARNA-Tijuca divides the northern and southern zones of the city of Rio de Janeiro, presenting significant Atlantic Forest vegetation cover and is a very important conservation unit for the protection of local fauna and flora diversity. In addition, the park also contributes to the quality of life of the inhabitants of the city of Rio de Janeiro and its visitors, as it provides green areas, recreation, tourism, and water supply (Freitas et al. 2006 ). The management plan for Tijuca National Park describes this conservation unit as very important for maintaining water sources, mitigating floods, regulating the local climate, reducing air and noise pollution, controlling erosion, and maintaining the natural landscape (ICMBIO 2008 ). Beliani et al. (2016) highlight that sector A of the park, the Tijuca Forest, suffered the most from human occupation and land use. However, recovery efforts dating back to 1886, during the imperial period of the country under Dom Pedro II (Almeida 2016 ), have allowed the Tijuca Forest to become an area of secondary vegetation in an advanced stage of succession. Nevertheless, PARNA-Tijuca is not entirely free from the problems that humans tend to cause in vegetated areas. Regarding anthropogenic impacts, Rocha et al. ( 2021 ) detected that on trails in Tijuca National Park, there is debris left behind by visitors, such as food packaging, glass bottles, candle wax, aluminum and glass materials that can harm wildlife and compromise visitor safety. Even vandalism is something that has been observed by these authors. Even vandalism was observed by these authors. On the other hand, Vilani and Souza (2017) emphasize that visitation to parks such as PARNA-Tijuca through ecotourism is not something that should be overlooked because it facilitates the learning process when combined with environmental education. This highlights the importance of the role of humans in terms of their impact on the environment and the need for environmental awareness to be a priority, given that Tijuca National Park is one of the main remnants of the Atlantic Forest biome in the state of Rio de Janeiro (Rocha et al. 2003 ). 2.2 Collection Points Two collections were carried out within specific sites in Tijuca National Park. The collection carried out in August 2021 comprised the Tijuca River section 1 and the Caveira River located within Tijuca National Park and the Tijuca River section 2 outside the conservation unit. The collection carried out in August 2019 only included the Tijuca section 1 and the Caveira River located within Tijuca National Park as collection sites. The Tijuca River section 1 was easily accessible and is located near the entrance to the Tijuca forest sector. The second section of the Tijuca River, which was outside the park, was not accessible from its banks. To reach this collection point, it was necessary to walk inside the river. The Caveira River, on the other hand, is located in a higher altitude portion of the park, with moderate accessibility. 2.3 Sampling Substrates present in river water, such as leaves or twigs, which had simulid larvae, were collected in 10 30x30 quadrats with an average distance of 5 meters between them. The Tijuca River -section 2 was collected in only 2 quadrats due to the length of this stretch and field conditions. This material was placed in labeled zip-lock plastic bags containing 70% ethyl alcohol for fixation and preservation of the larvae. During this stage, the collection protocol for immature simulids and other macroinvertebrates was completed in order to record the river data. Subsequently, the substrate was sorted ex situ , where, using metal tweezers, the larvae were removed and placed in Falcon-type bottles containing 70% ethyl alcohol. 2.4 Abiotic Variables Abiotic variables influence the distribution and life cycle of simulids, so they were determined in situ using portable equipment. The abiotic variables measured were water temperature, conductivity, pH, conductivity, Altitude, Light intensity and Geographic coordinates. Water velocity is another abiotic variable that was measured, but unlike the others, no specific equipment was used. It was determined by a method known as “Head Rod,” proposed by Wilm and Storey (1944). In this method, a 1-meter metal ruler is positioned so that its metric face is facing the bank, and the value indicated shows the depth of the river at that point. 2.5 Environmental Integrity Index of Rivers During the collection carried out in Tijuca National Park, each site underwent an assessment using the RCE (Riparian, Channel, and Environmental Inventory) protocol. This protocol makes it possible to determine the level of conservation of the river by calculating the environmental integrity index obtained by different scores that can be achieved after the river undergoes a questionnaire on the observation of different parameters, such as the state of preservation of the riparian forest zone, the structure of the channel, aquatic vegetation, the presence of debris, among others (Petersen 1992 ). The higher the river's score, the better its state of conservation and integrity, and it ends up being classified into five classes (I-V). 2.6 Identification of Larvae Species In the laboratory, simulid larvae were initially grouped according to their cephalic spot patterns, generating different morphotypes in a process called morphotyping. These spots are located on the cephalic region of simulid larvae and are used as criteria for identifying larval species. Only specimens containing histoblasts with mature respiratory filaments (visibly dark to the naked eye) were grouped into morphotypes. These morphological characteristics were visualized using a model 152b stereoscopic microscope (Digilab). Photographs of the larvae were obtained using a digital microscope (DW) with image capture software called hvcap version 1.2. The morphotypes were compared with specimens from the collection of the Aquatic Insects Laboratory of the National Museum of the Federal University of Rio de Janeiro (UFRJ). 2.7 Morphometry The photographs of the larvae obtained were submitted to image analysis software Cmeias-IT version 1.28, which allowed the measurement of the body sizes of the simulid larvae in mm²., using the methodology described in Figueiró et al. 2015 After this morphometric analysis to determine the body measurements of the larvae, a correlation was made between the body size of the last instar larvae of the species S . pertinax and S . ( Inaequalium ) sp. and the water quality conditions of the rivers. After calibrating the program with an object of known size, the cephalic region, propata, and body as a whole were the parts of the larvae on which morphometry was performed. 2.8 Statistical Analyses Canonical Correspondence Analysis (CCA) and Canonical Correlation Analysis (CCA) were performed using the Canoco program in order to correlate biotic and abiotic variables and determine which of the variables evaluated were significant. The statistical significance of the variables was tested by Monte Carlo permutations (5000). The Simpson diversity index was also measured. The normality test and the Wilcoxon signed rank test BioEstat 5.3 for non-parametric data were also performed in the program to verify whether there was a significant difference between the body sizes of the larvae collected in 2019 and 2021. 3 Results A total of 1,050 simulid larvae were collected in this study. The taxa found were: Simulium pertinax , Simulium (Inaequalium) sp ., Simulium (Psaroniocompsa) anamariae Vulcano, 1962, and Simulium (Psilopelmia) perflavum Roubaud, 1906. The species S. pertinax was found in all rivers and demonstrated the greatest abundance, with the Tijuca Stream (T1) in the August 2019 collection showing the highest number of larvae, with 400 individuals (Fig. 2 ). The results for the biodiversity indices can be seen in Table 1 . For the collection conducted in 2019, Caveira Stream was the site with the highest Simpson's diversity index and the lowest species dominance. Table 1 Results for the Simpson's Diversity and Dominance Indices from each site and collection. August 2019 Collection Sites Tijuca River – Inside (T1) Caveira River Dominance 0,9950 0,4567 Simpson's Diversity Index (1-D) 0,0049 0,5432 August 2021 Collection Sites Tijuca River – Inside (T1) Tijuca River – Outside (T2) Caveira River Dominance 0.5408 0.3626 0.5775 Simpson’s Diversity Index (1-D) 0.4592 0.6373 0.4224 The abiotic measurements taken in situ and the calculation of river water integrity are summarized in Table 2 . Conductivity and pH were the variables that differed the most for the stretch outside the conservation unit in the 2021 collection. Conductivity reached a maximum value of 44 µS/cm in the stretch outside the conservation unit, while the pH for the same site was in the range of 6.7. Table 2 – Set of abiotic variables measured at each site and collection August 2019 Collection Abiotic Variable Altitude (m) Conductivity (µS/cm) Temperature (°C) pH Luminosity (Lutz) Water velocity (m/s) Tijuca River – Inside (T1) 298 20 18,2 6,9 339,2 0,72 Caveira River 425 42 21,3 7,3 615,4 0,83 August 2021 Collection Abiotic Variable Altitude (m) Conductivity (µS/cm) Temperature (°C) pH Luminosity (Lutz) Water velocity (m/s) Tijuca River – Inside (T1) 223 22 22,3 7,3 983,2 0,84 Tijuca River – Outside (T2) 215 44 23,6 6,7 641,6 0,70 Caveira River 493 25 21,1 7,1 671,2 0,80 The abiotic and biotic data from the two years of collection were then grouped and applied to a Canonical Correspondence Analysis (CCA) (Fig. 3 ), showing that S. pertinax was the only species negatively influenced by most environmental factors. It is important to highlight that only the abiotic variables of conductivity, altitude, and position within the stream (middle or margin) were significant. S. anamariae had a strong positive correlation with water conductivity. S. (Inaequalium) sp . was positively influenced by the significant variables of altitude and associated with the middle of the water courses. The species S. anamariae had a strong positive correlation with water conductivity., while S. (Inaequalium) sp . was positively correlated with the altitude and was associated with the middle of the water courses. A nonparametric Wilcoxon signed-rank test was performed, which showed that for both S. pertinax and S. (Inaequalium) sp ., there was a significant difference in larval body measurements between 2019 and 2021 (p-value < 0.05). In the next step, canonical correspondence analysis (CCA) was performed for the morphometric measurements of the body, head/cephalic area, and protorax leg for S. pertinax and S. (Inaequalium) sp . to investigate the influence of environmental variables on them (Fig. 4 ). Only light and altitude were found to be significant. The body, head, and prostrate measurements of the species S. pertinax were strongly negatively influenced by both altitude and light. The body measurements of S. (Inaequalium) sp . were positively associated with altitude, and head/cephalic region measurements were positively influenced by light. These two species appear to have divergent morphological responses to body size as a function of environmental variables. To deepen the interpretations regarding the relationships between the body, cephalic/head region, and protorax leg of larvae and different environmental variables, a canonical correlation was performed. The results of this analysis showed that the correlations between the two groups (abiotic variables and morphometric measurements) were highly significant, with a canonical R of 0.9601 and a highly significant p-value of 0.0001 in some of the correlations. In Table 4 it is possible to see the canonical correlation matrix, where there are 16 correlations that are significant. Table 4 Canonical Correlations for Larval Morphometry Correlations Xi vs. Yi Variable Body S. (Inaequalium) sp . Protorax leg S. (Inaequalium) sp. Head S. (Inaequalium) sp. Body S. pertinax Protorax leg S. pertinax Head S. pertinax Depth -0.012 ns -0.127 ns 0.178 ns -0.041 ns -0.069 ns -0.014 ns Water velocity 0.017 ns 0.026 ns 0.189 ns -0.142 ns -0.182 ns -0.121 ns pH -0.455 (p < 0.01) -0.246 ns 0.563 (p < 0.01) -0.554 (p < 0.01) -0.613 (p < 0.01) -0.538 (p < 0.01) Temperature 0.247 ns 0.401 (p < 0.01) 0.851 (p < 0.01) -0.350 (p < 0.05) -0.409 (p < 0.01) -0.354 (p < 0.05) Conductivity 0.368 (p < 0.05) 0.357 (p < 0.05) 0.489 (p < 0.01) -0.070 ns -0.079 ns -0.101 ns Altitude -0.233 ns -0.109 ns 0.221 ns -0.452 (p < 0.01) − 0.445 (p < 0.01) − 0.445 (p < 0.01) 4 Discussion The species S. pertinax showed a relative abundance of 59% in 2019 and 16% in 2021. In the literature, studies such as those by Menzel et al. (2919) have also investigated the distribution and abundance of this species. They verified blackflies species in the Ijuí River Basin, Rio Grande do Sul and found that S. pertinax was the most abundant, representing 61.35% of the total. Couceiro et al. ( 2014 ) found that for streams in Rio Grande do Sul, S. pertinax was present in over 50% of the collection sites and that its abundance was higher in streams with high water pH and electrical conductivity. Abiotic variables are important parameters that affect the prevalence of simuliid species. Each simuliid species prefers different sets of physicochemical parameters (Rabha et al. 2013 ). Buitrago-Guacaneme et al. ( 2018 ) also emphasize the need for further studies correlating aquatic biota with environmental factors. Abiotic factors may also be responsible for morphological changes in simulid larvae: for example, larvae that colonize habitats with more turbulent currents and higher altitudes may have darker head capsules than those living in lower locations with lower water flow. The mechanical stress of the current and the greater incidence of radiation at higher altitudes are responsible for this color change (Car and Lechthaler 2006 ). In relation to water velocity, the 2021 collection in the outside section 2 , showed 0.63 m/s and 0.77 m/s water velocities, and although these values are below the velocities found in other quadrats, other sites presented values lower than these. Bertazo and Figueiró (2012) observed that blackfly larvae can have different preferences for current velocity gradients in Aracruz, Espírito Santo. The species Simulium brachycladum Lutz and Pinto 1932, showed an association with medium velocities, while the species Simulium subpallidum Lutz, 1909, was associated with different velocity values, both medium and high, showing a more generalist behavior. S. (Psaroniocompsa) sp. was present in lower velocities ranging from 0.44 to 0.57 m/s up to 0.62 to 0.81 m/s. The results of the morphometric analysis showed that the larger larvae occurred in the 2021 collection, with 5.84 mm 2 for the average body of S. pertinax in Tijuca Stream section 2 , 0.11 mm 2 for the propata of S. (Inaequalium) sp . in Caveira Stream, and 0.73 mm 2 for the cephalic region in Tijuca Stream section 2 . Zhang and Malmqvist ( 1996 ) observed that larger larvae were colonizing slow-flowing habitats, and the largest larval body size recorded was 8.92 mm for the species Prosimulium rufum Frey, 1935. Furthermore, combining physicochemical analyses with biological data using macroinvertebrates can provide greater precision in identifying anthropogenic impacts on water resources, as physicochemical data reflect only the conditions at the exact moment of measurement, while macroinvertebrate assessment can provide data on stress events over time. It is also much more cost-effective than physicochemical methodologies, providing a useful alternative for locations lacking significant resources (Rai et al. 2020 ). 5 Conclusion The results indicate overall larger body sizes of S. pertinax and S. (Inaequalium) sp . outside Tijuca National Park than those of the larvae found inside. Furthermore, there were significant differences in the body sizes of the two species between 2019 and 2021 for the rivers located within the park. There was a reduction in the relative abundance of S. pertinax when comparing the two years studied. Currently, few studies approach the correlation of the body size of simulid larvae with river water integrity indices, and more in-depth analyses may be necessary to confirm the relationship between the two species and whether they can indeed be used as biological indicators. Declarations Acknowledgements We thank the aquatic dipteran laboratory of the entomology department of the National Museum/UFRJ for their help in identifying the specimens. Authors' contributions TMMSM, TD and RF were responsible for the project’s conceptualization, management, and supervision. TMMSM conducted field collections and laboratory analyses. LHGA and IKLM conducted taxonomic identification. TMMSM and RF performed statistical analyses. BAV, TD, IKLM and AAS wrote the manuscript; all authors contributed to its review and editing. Funding FAPERJ and CAPES. Data availability All data generated during this study are available from the corresponding author. 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Biological Journal of the Linnean Society 59:261-280 Supplementary Files Appendix.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":1202957,"visible":true,"origin":"","legend":"\u003cp\u003eMap with collection points, made with QGIS 3.34. The collection point can be seen outside the boundaries of the conservation unit, delimited by the yellow line. The other two collection points are located within the Tijuca National Park.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8205131/v1/b049b8f2469d0a26a28f20b7.png"},{"id":99318790,"identity":"50fcb27e-166e-49e5-bd62-c83b6eb5b4f5","added_by":"auto","created_at":"2025-12-31 16:34:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":226344,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of individuals of specific species for each site. The graph illustrates the respective species found in the August 2019 collection as a function of the number of larvae. It is possible to observe the greater abundance of \u003cem\u003eS. pertinax\u003c/em\u003e in the Tijuca River, section 1. In 2021, a greater number of unidentified larvae were obtained. There is also a reduction in the species \u003cem\u003eS. pertinax\u003c/em\u003e for the same collection sites as the previous year.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8205131/v1/abb374d5f4f9419719cf8463.png"},{"id":99222639,"identity":"3b79a145-01d0-4547-a9b2-e40603af1028","added_by":"auto","created_at":"2025-12-30 09:56:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":55239,"visible":true,"origin":"","legend":"\u003cp\u003eCCA analysis for abiotic variables and species composition (Conductivity, Altitude and position within the stream).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8205131/v1/cc20a46ae1186298f727332d.png"},{"id":99222635,"identity":"a53ed903-e291-4fff-b4c7-eb9bb4bdac94","added_by":"auto","created_at":"2025-12-30 09:56:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":52754,"visible":true,"origin":"","legend":"\u003cp\u003eCCA analysis for morphometric data (Only light and altitude were significant).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8205131/v1/65148853e9a983f9fa8ab425.png"},{"id":105565190,"identity":"f2529f5d-0682-4321-b796-531ee80ee794","added_by":"auto","created_at":"2026-03-27 12:52:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2237826,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8205131/v1/b9958eb5-f856-4c47-953d-be94daac6955.pdf"},{"id":99318410,"identity":"dfd832fa-22b1-4ddd-bcd1-0650076967a0","added_by":"auto","created_at":"2025-12-31 16:33:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14670,"visible":true,"origin":"","legend":"","description":"","filename":"Appendix.docx","url":"https://assets-eu.researchsquare.com/files/rs-8205131/v1/73e1ad545bf68e6ab58375b7.docx"}],"financialInterests":"","formattedTitle":"Morphometric analysis of black fly larvae (Diptera: Simuliidae) in Tijuca National Park, Rio de Janeiro, Brazil: A potential tool for biomonitoring aquatic environments","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eWater is an essential natural resource not only for humans, but for all living beings, harboring enormous biodiversity in aquatic ecosystems. Surface freshwater, found in rivers and lakes, represents only about 0.01% of the total water on Earth (Kuhlmann et al. 2019). However, this ecosystem can be impacted by anthropogenic actions that compromise the quality of these water bodies, such as deforestation of riparian forests, pollution from industry, households and agriculture, and the introduction of exotic species, among others (Baptista, et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to CONAMA Resolution 001 of 23 January 1986, an environmental impact can be defined as \u0026ldquo;any change in the physical, chemical and biological properties of the environment resulting from human activities that directly or indirectly affects [...] the quality of environmental resources\u0026rdquo;. With this, it is increasingly apparent that studying the environmental impacts caused by human activities on water bodies is vital for their management and administration, which can ensure more effective decision-making in their protection or recovery (Jacinto-Junior and Lucena \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To this end, constant biomonitoring is necessary to detect these environmental impacts in their early stages, since once serious water degradation has been reached, it becomes difficult to reverse the scenario (Baptista \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe assessment of biotic communities for the purpose of investigating ecosystem conservation through environmental monitoring and bioindicators has proven to be an alternative for detecting impacts resulting from human activity (Docile and Figueir\u0026oacute; \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Silva and Viana 2021). Among the different bioindicator organisms, the order Diptera is considered a good environmental indicator because they are abundant in lotic or lentic aquatic environments and can tolerate high concentrations of organic matter in waters with low oxygenation (Lima et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Included in this order are insects of the Simullidae family, also known as simulids. The larvae of these insects are filter feeders, and their diet consists of organic matter dissolved in the water column (Bernotiene 2006).\u003c/p\u003e \u003cp\u003eAmong the Diptera, simuliids, also known as black flies, are a dipteran family that breeds in lotic systems (Hamada and McCreadie 2002, Landeiro et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Figueir\u0026oacute; et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Menzel et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Lima-De-Sousa et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), feeding on suspended organic matter in the water column. There are studies that mention simuliids as environmental bioindicators, meaning they can be used as a tool to assess the ecological status of watercourses in urbanized areas (Couceiro et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Ciadamidaro et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This occurs because anthropized sites may favour certain species (Coppo and Lopes 2010; Docile et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Costa et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The literature reports that some species, such as \u003cem\u003eSimulium pertinax\u003c/em\u003e Kollar, 1832, can develop in aquatic environments impacted by varying degrees of pollution (Strieder et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). One of the influences that an impacted environment can exert is on the body size of simulid larvae. Ciborowski and his collaborators (1997) observed that the availability of organic matter in water can influence a variation in the body size of simulid larvae, while Figueir\u0026oacute; et al (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and Pinto (2024) observed the phenotypic plasticity of these organisms among different microhabitats.\u003c/p\u003e \u003cp\u003eThis study considers the premise that water bodies located within Tijuca National Park will have a better conservation status than those located outside the park, since it is a fully protected conservation unit (ICMBIO et al. 2008). Collection sites were therefore selected both inside and outside this conservation unit. Tijuca National Park has several water sources that are used to supply the homes surrounding the park and are also used for recreational purposes (Silva et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Within the park, there is also a water treatment plant called Afonso Viseu, which is responsible for part of the drinking water supply for Alto da Boa Vista (CEDAE \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), highlighting the importance of conserving the water resources of Tijuca National Park and the fundamental role of environmental monitoring in ensuring the monitoring and protection of water bodies. Therefore, this study aims to evaluate how the conservation status of water bodies inside and outside Tijuca National Park influences the abundance and body size of Simuliidae larvae, contributing to the understanding of how environmental quality affects these bioindicator organisms.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Study area\u003c/h2\u003e \u003cp\u003eTijuca National Park (PARNA-Tijuca), created in 1961 initially under the name Rio de Janeiro National Park (BRAZIL 1961), is a fully protected conservation area under Law No. 9,985 of July 18, 2000, covering an area of 3, 953 hectares located between parallels 22\u0026deg;55' South and 23\u0026deg;00' South and meridians 43\u0026deg;12' West and 43\u0026deg;19' West, in the south-central part of the state of Rio de Janeiro (ICMBIO \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). According to Souza (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), on a global scale, PARNA-Tijuca is one of the largest replanted forests located in an urban area. PARNA-Tijuca is divided into four main areas that can be differentiated according to their state of conservation, land use, and environmental characteristics. These sectors are called: Serra da Carioca, Floresta da Tijuca, Pedra Bonita/Pedra da G\u0026aacute;vea, and Pretos-Forros/Covanca (Zago et al. 2020).\u003c/p\u003e \u003cp\u003eThe location of PARNA-Tijuca divides the northern and southern zones of the city of Rio de Janeiro, presenting significant Atlantic Forest vegetation cover and is a very important conservation unit for the protection of local fauna and flora diversity. In addition, the park also contributes to the quality of life of the inhabitants of the city of Rio de Janeiro and its visitors, as it provides green areas, recreation, tourism, and water supply (Freitas et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The management plan for Tijuca National Park describes this conservation unit as very important for maintaining water sources, mitigating floods, regulating the local climate, reducing air and noise pollution, controlling erosion, and maintaining the natural landscape (ICMBIO \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBeliani et al. (2016) highlight that sector A of the park, the Tijuca Forest, suffered the most from human occupation and land use. However, recovery efforts dating back to 1886, during the imperial period of the country under Dom Pedro II (Almeida \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), have allowed the Tijuca Forest to become an area of secondary vegetation in an advanced stage of succession.\u003c/p\u003e \u003cp\u003eNevertheless, PARNA-Tijuca is not entirely free from the problems that humans tend to cause in vegetated areas. Regarding anthropogenic impacts, Rocha et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) detected that on trails in Tijuca National Park, there is debris left behind by visitors, such as food packaging, glass bottles, candle wax, aluminum and glass materials that can harm wildlife and compromise visitor safety.\u003c/p\u003e \u003cp\u003eEven vandalism is something that has been observed by these authors. Even vandalism was observed by these authors. On the other hand, Vilani and Souza (2017) emphasize that visitation to parks such as PARNA-Tijuca through ecotourism is not something that should be overlooked because it facilitates the learning process when combined with environmental education.\u003c/p\u003e \u003cp\u003eThis highlights the importance of the role of humans in terms of their impact on the environment and the need for environmental awareness to be a priority, given that Tijuca National Park is one of the main remnants of the Atlantic Forest biome in the state of Rio de Janeiro (Rocha et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Collection Points\u003c/h2\u003e \u003cp\u003eTwo collections were carried out within specific sites in Tijuca National Park. The collection carried out in August 2021 comprised the Tijuca River section \u003cspan refid=\"Sec1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and the Caveira River located within Tijuca National Park and the Tijuca River section \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003e2\u003c/span\u003e outside the conservation unit. The collection carried out in August 2019 only included the Tijuca section \u003cspan refid=\"Sec1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and the Caveira River located within Tijuca National Park as collection sites.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Tijuca River section \u003cspan refid=\"Sec1\" class=\"InternalRef\"\u003e1\u003c/span\u003e was easily accessible and is located near the entrance to the Tijuca forest sector. The second section of the Tijuca River, which was outside the park, was not accessible from its banks. To reach this collection point, it was necessary to walk inside the river. The Caveira River, on the other hand, is located in a higher altitude portion of the park, with moderate accessibility.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Sampling\u003c/h2\u003e \u003cp\u003eSubstrates present in river water, such as leaves or twigs, which had simulid larvae, were collected in 10 30x30 quadrats with an average distance of 5 meters between them. The Tijuca River -section \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003e2\u003c/span\u003e was collected in only 2 quadrats due to the length of this stretch and field conditions. This material was placed in labeled zip-lock plastic bags containing 70% ethyl alcohol for fixation and preservation of the larvae. During this stage, the collection protocol for immature simulids and other macroinvertebrates was completed in order to record the river data. Subsequently, the substrate was sorted \u003cem\u003eex situ\u003c/em\u003e, where, using metal tweezers, the larvae were removed and placed in Falcon-type bottles containing 70% ethyl alcohol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Abiotic Variables\u003c/h2\u003e \u003cp\u003eAbiotic variables influence the distribution and life cycle of simulids, so they were determined \u003cem\u003ein situ\u003c/em\u003e using portable equipment. The abiotic variables measured were water temperature, conductivity, pH, conductivity, Altitude, Light intensity and Geographic coordinates. Water velocity is another abiotic variable that was measured, but unlike the others, no specific equipment was used. It was determined by a method known as \u0026ldquo;Head Rod,\u0026rdquo; proposed by Wilm and Storey (1944). In this method, a 1-meter metal ruler is positioned so that its metric face is facing the bank, and the value indicated shows the depth of the river at that point.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Environmental Integrity Index of Rivers\u003c/h2\u003e \u003cp\u003eDuring the collection carried out in Tijuca National Park, each site underwent an assessment using the RCE (Riparian, Channel, and Environmental Inventory) protocol. This protocol makes it possible to determine the level of conservation of the river by calculating the environmental integrity index obtained by different scores that can be achieved after the river undergoes a questionnaire on the observation of different parameters, such as the state of preservation of the riparian forest zone, the structure of the channel, aquatic vegetation, the presence of debris, among others (Petersen \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). The higher the river's score, the better its state of conservation and integrity, and it ends up being classified into five classes (I-V).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Identification of Larvae Species\u003c/h2\u003e \u003cp\u003eIn the laboratory, simulid larvae were initially grouped according to their cephalic spot patterns, generating different morphotypes in a process called morphotyping. These spots are located on the cephalic region of simulid larvae and are used as criteria for identifying larval species. Only specimens containing histoblasts with mature respiratory filaments (visibly dark to the naked eye) were grouped into morphotypes. These morphological characteristics were visualized using a model 152b stereoscopic microscope (Digilab). Photographs of the larvae were obtained using a digital microscope (DW) with image capture software called hvcap version 1.2. The morphotypes were compared with specimens from the collection of the Aquatic Insects Laboratory of the National Museum of the Federal University of Rio de Janeiro (UFRJ).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Morphometry\u003c/h2\u003e \u003cp\u003eThe photographs of the larvae obtained were submitted to image analysis software Cmeias-IT version 1.28, which allowed the measurement of the body sizes of the simulid larvae in mm\u0026sup2;., using the methodology described in Figueir\u0026oacute; et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e After this morphometric analysis to determine the body measurements of the larvae, a correlation was made between the body size of the last instar larvae of the species \u003cem\u003eS\u003c/em\u003e. \u003cem\u003epertinax\u003c/em\u003e and \u003cem\u003eS\u003c/em\u003e. (\u003cem\u003eInaequalium\u003c/em\u003e) sp. and the water quality conditions of the rivers. After calibrating the program with an object of known size, the cephalic region, propata, and body as a whole were the parts of the larvae on which morphometry was performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical Analyses\u003c/h2\u003e \u003cp\u003eCanonical Correspondence Analysis (CCA) and Canonical Correlation Analysis (CCA) were performed using the Canoco program in order to correlate biotic and abiotic variables and determine which of the variables evaluated were significant. The statistical significance of the variables was tested by Monte Carlo permutations (5000). The Simpson diversity index was also measured. The normality test and the Wilcoxon signed rank test BioEstat 5.3 for non-parametric data were also performed in the program to verify whether there was a significant difference between the body sizes of the larvae collected in 2019 and 2021.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003eA total of 1,050 simulid larvae were collected in this study. The taxa found were: \u003cem\u003eSimulium pertinax\u003c/em\u003e, \u003cem\u003eSimulium (Inaequalium) sp\u003c/em\u003e., \u003cem\u003eSimulium (Psaroniocompsa) anamariae\u003c/em\u003e Vulcano, 1962, and \u003cem\u003eSimulium (Psilopelmia) perflavum\u003c/em\u003e Roubaud, 1906. The species \u003cem\u003eS. pertinax\u003c/em\u003e was found in all rivers and demonstrated the greatest abundance, with the Tijuca Stream (T1) in the August 2019 collection showing the highest number of larvae, with 400 individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results for the biodiversity indices can be seen in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. For the collection conducted in 2019, Caveira Stream was the site with the highest Simpson's diversity index and the lowest species dominance.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults for the Simpson's Diversity and Dominance Indices from each site and collection.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eAugust 2019 Collection\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSites\u003c/p\u003e \u003cp\u003e\u003cem\u003eTijuca River \u0026ndash; Inside (T1)\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eCaveira River\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDominance\u003c/p\u003e \u003cp\u003e0,9950\u003c/p\u003e \u003cp\u003e0,4567\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSimpson's Diversity Index (1-D)\u003c/p\u003e \u003cp\u003e0,0049\u003c/p\u003e \u003cp\u003e0,5432\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eAugust 2021 Collection\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSites\u003c/p\u003e \u003cp\u003e\u003cem\u003eTijuca River \u0026ndash; Inside (T1)\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eTijuca River \u0026ndash; Outside (T2)\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eCaveira River\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDominance\u003c/p\u003e \u003cp\u003e0.5408\u003c/p\u003e \u003cp\u003e0.3626\u003c/p\u003e \u003cp\u003e0.5775\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSimpson\u0026rsquo;s Diversity Index (1-D)\u003c/p\u003e \u003cp\u003e0.4592\u003c/p\u003e \u003cp\u003e0.6373\u003c/p\u003e \u003cp\u003e0.4224\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe abiotic measurements taken \u003cem\u003ein situ\u003c/em\u003e and the calculation of river water integrity are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Conductivity and pH were the variables that differed the most for the stretch outside the conservation unit in the 2021 collection. Conductivity reached a maximum value of 44 \u0026micro;S/cm in the stretch outside the conservation unit, while the pH for the same site was in the range of 6.7.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u0026ndash; Set of abiotic variables measured at each site and collection\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eAugust 2019 Collection\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbiotic Variable\u003c/p\u003e \u003cp\u003eAltitude (m)\u003c/p\u003e \u003cp\u003eConductivity (\u0026micro;S/cm)\u003c/p\u003e \u003cp\u003eTemperature (\u0026deg;C)\u003c/p\u003e \u003cp\u003epH\u003c/p\u003e \u003cp\u003eLuminosity (Lutz)\u003c/p\u003e \u003cp\u003eWater velocity (m/s)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTijuca River \u0026ndash; Inside (T1)\u003c/em\u003e\u003c/p\u003e \u003cp\u003e298\u003c/p\u003e \u003cp\u003e20\u003c/p\u003e \u003cp\u003e18,2\u003c/p\u003e \u003cp\u003e6,9\u003c/p\u003e \u003cp\u003e339,2\u003c/p\u003e \u003cp\u003e\u003cem\u003e0,72\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaveira River\u003c/em\u003e\u003c/p\u003e \u003cp\u003e425\u003c/p\u003e \u003cp\u003e42\u003c/p\u003e \u003cp\u003e21,3\u003c/p\u003e \u003cp\u003e7,3\u003c/p\u003e \u003cp\u003e615,4\u003c/p\u003e \u003cp\u003e\u003cem\u003e0,83\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eAugust 2021 Collection\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbiotic Variable\u003c/p\u003e \u003cp\u003eAltitude (m)\u003c/p\u003e \u003cp\u003eConductivity (\u0026micro;S/cm)\u003c/p\u003e \u003cp\u003eTemperature (\u0026deg;C)\u003c/p\u003e \u003cp\u003epH\u003c/p\u003e \u003cp\u003eLuminosity (Lutz)\u003c/p\u003e \u003cp\u003eWater velocity (m/s)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTijuca River \u0026ndash; Inside (T1)\u003c/em\u003e\u003c/p\u003e \u003cp\u003e223\u003c/p\u003e \u003cp\u003e22\u003c/p\u003e \u003cp\u003e22,3\u003c/p\u003e \u003cp\u003e7,3\u003c/p\u003e \u003cp\u003e983,2\u003c/p\u003e \u003cp\u003e0,84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cem\u003eTijuca River \u0026ndash; Outside (T2)\u003c/em\u003e\u003c/p\u003e \u003cp\u003e215\u003c/p\u003e \u003cp\u003e44\u003c/p\u003e \u003cp\u003e23,6\u003c/p\u003e \u003cp\u003e6,7\u003c/p\u003e \u003cp\u003e641,6\u003c/p\u003e \u003cp\u003e0,70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaveira River\u003c/em\u003e\u003c/p\u003e \u003cp\u003e493\u003c/p\u003e \u003cp\u003e25\u003c/p\u003e \u003cp\u003e21,1\u003c/p\u003e \u003cp\u003e7,1\u003c/p\u003e \u003cp\u003e671,2\u003c/p\u003e \u003cp\u003e0,80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe abiotic and biotic data from the two years of collection were then grouped and applied to a Canonical Correspondence Analysis (CCA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), showing that \u003cem\u003eS. pertinax\u003c/em\u003e was the only species negatively influenced by most environmental factors. It is important to highlight that only the abiotic variables of conductivity, altitude, and position within the stream (middle or margin) were significant. \u003cem\u003eS. anamariae\u003c/em\u003e had a strong positive correlation with water conductivity. \u003cem\u003eS. (Inaequalium) sp\u003c/em\u003e. was positively influenced by the significant variables of altitude and associated with the middle of the water courses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe species \u003cem\u003eS. anamariae\u003c/em\u003e had a strong positive correlation with water conductivity., while \u003cem\u003eS. (Inaequalium) sp\u003c/em\u003e. was positively correlated with the altitude and was associated with the middle of the water courses.\u003c/p\u003e \u003cp\u003eA nonparametric Wilcoxon signed-rank test was performed, which showed that for both \u003cem\u003eS. pertinax\u003c/em\u003e and \u003cem\u003eS. (Inaequalium) sp\u003c/em\u003e., there was a significant difference in larval body measurements between 2019 and 2021 (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the next step, canonical correspondence analysis (CCA) was performed for the morphometric measurements of the body, head/cephalic area, and protorax leg for \u003cem\u003eS. pertinax\u003c/em\u003e and \u003cem\u003eS. (Inaequalium) sp\u003c/em\u003e. to investigate the influence of environmental variables on them (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOnly light and altitude were found to be significant. The body, head, and prostrate measurements of the species \u003cem\u003eS. pertinax\u003c/em\u003e were strongly negatively influenced by both altitude and light. The body measurements of \u003cem\u003eS. (Inaequalium) sp\u003c/em\u003e. were positively associated with altitude, and head/cephalic region measurements were positively influenced by light. These two species appear to have divergent morphological responses to body size as a function of environmental variables.\u003c/p\u003e \u003cp\u003eTo deepen the interpretations regarding the relationships between the body, cephalic/head region, and protorax leg of larvae and different environmental variables, a canonical correlation was performed. The results of this analysis showed that the correlations between the two groups (abiotic variables and morphometric measurements) were highly significant, with a canonical R of 0.9601 and a highly significant p-value of 0.0001 in some of the correlations. In Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e it is possible to see the canonical correlation matrix, where there are 16 correlations that are significant.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCanonical Correlations for Larval Morphometry\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eCorrelations Xi vs. Yi\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBody \u003cem\u003eS. (Inaequalium) sp\u003c/em\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProtorax leg S. \u003cem\u003e(Inaequalium) sp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHead S. \u003cem\u003e(Inaequalium) sp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBody \u003cem\u003eS. pertinax\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eProtorax leg \u003cem\u003eS. pertinax\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHead \u003cem\u003eS. pertinax\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDepth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.012 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.127 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.178 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0.041 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.069 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.014 ns\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater velocity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.017 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.026 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.189 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0.142 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.182 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.121 ns\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.455\u003c/p\u003e \u003cp\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.246 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.563\u003c/p\u003e \u003cp\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0.554\u003c/p\u003e \u003cp\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.613\u003c/p\u003e \u003cp\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.538\u003c/p\u003e \u003cp\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.247 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.401\u003c/p\u003e \u003cp\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.851\u003c/p\u003e \u003cp\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0.350 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.409\u003c/p\u003e \u003cp\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.354 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConductivity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.368\u003c/p\u003e \u003cp\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.357\u003c/p\u003e \u003cp\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.489\u003c/p\u003e \u003cp\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0.070 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.079 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.101 ns\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAltitude\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.233 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.109 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.221 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0.452\u003c/p\u003e \u003cp\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;0.445 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;0.445 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe species \u003cem\u003eS. pertinax\u003c/em\u003e showed a relative abundance of 59% in 2019 and 16% in 2021. In the literature, studies such as those by Menzel et al. (2919) have also investigated the distribution and abundance of this species. They verified blackflies species in the Iju\u0026iacute; River Basin, Rio Grande do Sul and found that \u003cem\u003eS. pertinax\u003c/em\u003e was the most abundant, representing 61.35% of the total. Couceiro et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) found that for streams in Rio Grande do Sul, \u003cem\u003eS. pertinax\u003c/em\u003e was present in over 50% of the collection sites and that its abundance was higher in streams with high water pH and electrical conductivity.\u003c/p\u003e \u003cp\u003eAbiotic variables are important parameters that affect the prevalence of simuliid species. Each simuliid species prefers different sets of physicochemical parameters (Rabha et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Buitrago-Guacaneme et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) also emphasize the need for further studies correlating aquatic biota with environmental factors. Abiotic factors may also be responsible for morphological changes in simulid larvae: for example, larvae that colonize habitats with more turbulent currents and higher altitudes may have darker head capsules than those living in lower locations with lower water flow. The mechanical stress of the current and the greater incidence of radiation at higher altitudes are responsible for this color change (Car and Lechthaler \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn relation to water velocity, the 2021 collection in the outside section \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, showed 0.63 m/s and 0.77 m/s water velocities, and although these values are below the velocities found in other quadrats, other sites presented values lower than these. Bertazo and Figueir\u0026oacute; (2012) observed that blackfly larvae can have different preferences for current velocity gradients in Aracruz, Esp\u0026iacute;rito Santo. The species \u003cem\u003eSimulium brachycladum\u003c/em\u003e Lutz and Pinto 1932, showed an association with medium velocities, while the species \u003cem\u003eSimulium subpallidum\u003c/em\u003e Lutz, 1909, was associated with different velocity values, both medium and high, showing a more generalist behavior. \u003cem\u003eS. (Psaroniocompsa)\u003c/em\u003e sp. was present in lower velocities ranging from 0.44 to 0.57 m/s up to 0.62 to 0.81 m/s.\u003c/p\u003e \u003cp\u003eThe results of the morphometric analysis showed that the larger larvae occurred in the 2021 collection, with 5.84 mm\u003csup\u003e2\u003c/sup\u003e for the average body of \u003cem\u003eS. pertinax\u003c/em\u003e in Tijuca Stream section \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, 0.11 mm\u003csup\u003e2\u003c/sup\u003e for the propata of \u003cem\u003eS. (Inaequalium) sp\u003c/em\u003e. in Caveira Stream, and 0.73 mm\u003csup\u003e2\u003c/sup\u003e for the cephalic region in Tijuca Stream section \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Zhang and Malmqvist (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) observed that larger larvae were colonizing slow-flowing habitats, and the largest larval body size recorded was 8.92 mm for the species \u003cem\u003eProsimulium rufum\u003c/em\u003e Frey, 1935.\u003c/p\u003e \u003cp\u003eFurthermore, combining physicochemical analyses with biological data using macroinvertebrates can provide greater precision in identifying anthropogenic impacts on water resources, as physicochemical data reflect only the conditions at the exact moment of measurement, while macroinvertebrate assessment can provide data on stress events over time. It is also much more cost-effective than physicochemical methodologies, providing a useful alternative for locations lacking significant resources (Rai et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThe results indicate overall larger body sizes of \u003cem\u003eS. pertinax\u003c/em\u003e and \u003cem\u003eS. (Inaequalium) sp\u003c/em\u003e. outside Tijuca National Park than those of the larvae found inside. Furthermore, there were significant differences in the body sizes of the two species between 2019 and 2021 for the rivers located within the park. There was a reduction in the relative abundance of \u003cem\u003eS. pertinax\u003c/em\u003e when comparing the two years studied. Currently, few studies approach the correlation of the body size of simulid larvae with river water integrity indices, and more in-depth analyses may be necessary to confirm the relationship between the two species and whether they can indeed be used as biological indicators.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the aquatic dipteran laboratory of the entomology department of the National Museum/UFRJ for their help in identifying the specimens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTMMSM, TD and RF were responsible for the project\u0026rsquo;s conceptualization, management, and supervision. TMMSM conducted field collections and laboratory analyses. LHGA and IKLM conducted taxonomic identification. TMMSM and RF performed statistical analyses. BAV, TD, IKLM and AAS wrote the manuscript; all authors contributed to its review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFAPERJ and CAPES.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All data generated during this study are available from the corresponding author. \u0026nbsp;No additional datasets were generated or deposited in external repositories.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot aplicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest/Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest or competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlmeida DS (2016) Hist\u0026oacute;rico e tend\u0026ecirc;ncias atuais da recupera\u0026ccedil;\u0026atilde;o ambiental. 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RIMA, S\u0026atilde;o Carlos, 134 pp\u003c/li\u003e\n\u003cli\u003eRocha MB, Gomes SBV, Rocha RO, Passei MG (2021) Identifica\u0026ccedil;\u0026atilde;o de impactos ambientais relacionados a visita\u0026ccedil;\u0026atilde;o p\u0026uacute;blica no Parque Nacional da Tijuca: O caso da trilha do estudante. Revista Tecnologia e Sociedade 17:1-19. https://doi.org/10.3895/rts.v16n39.8966 \u003c/li\u003e\n\u003cli\u003eSilva MS, Raposo MR, Pereira W, Bueno C (2017) Determina\u0026ccedil;\u0026atilde;o de radionucl\u0026iacute;deos e outros par\u0026acirc;metros na \u0026aacute;gua das fontes do Parque Nacional da Tijuca, Rio de Janeiro. Revista Semioses 11:12-20\u003c/li\u003e\n\u003cli\u003eSilva MVR, Vianam DSF (2021) Biomonitoramento do potencial do risco genot\u0026oacute;xico e an\u0026aacute;lise f\u0026iacute;sico-qu\u0026iacute;mica da \u0026aacute;gua do Balne\u0026aacute;rio Veneza no munic\u0026iacute;pio de Caxias-MA. 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Biological Journal of the Linnean Society 59:261-280\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Simulids, Morphometric analysis, Biomonitoring, Environmental indicators","lastPublishedDoi":"10.21203/rs.3.rs-8205131/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8205131/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSimulids are dipteran insects that colonize freshwater environments in their immature life stages. This study aims to compare the body size of Simulid larvae of the species \u003cem\u003eSimulium pertinax\u003c/em\u003e and \u003cem\u003eSimulium (Inaequalium) sp.\u003c/em\u003e collected in waterways inside and outside of Tijuca National Park. Collections were made in 2019 and 2021. Abiotic variables were verified in situ using specific equipment, and a protocol was also applied to assess the integrity of the river waters in each river. A total of 1,311 larvae were collected and found to be the following species: S\u003cem\u003eimulium pertinax, Simulium (Inaequalium) sp., Simulium (Psaroniocompsa) sp., Simulium (Psaroniocompsa) anamariae, and Simulium (Psilopelmia) perflavum\u003c/em\u003e. The average body, head, and protorax leg sizes of the larvae were larger in the outer section of the Tijuca River than in the inner section. The Wilcoxon test found a significant difference (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the body size of \u003cem\u003eS. pertinax\u003c/em\u003e and \u003cem\u003eS. (Inaequalium) sp.\u003c/em\u003e compared to 2019 and 2021. The CCA also showed that the body, head, and propata sizes of \u003cem\u003eS. pertinax\u003c/em\u003e were influenced by river brightness and altitude. The correlations between the abiotic variables and morphometric measurements were highly significant. As there are still few studies linking the morphometry of blackfly larvae to river water integrity, this work stands out for advancing in this field. Our results reinforce the potential of these organisms as biological indicators and point to new pathways for the biomonitoring of aquatic environments.\u003c/p\u003e","manuscriptTitle":"Morphometric analysis of black fly larvae (Diptera: Simuliidae) in Tijuca National Park, Rio de Janeiro, Brazil: A potential tool for biomonitoring aquatic environments","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-30 09:56:22","doi":"10.21203/rs.3.rs-8205131/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c18bd5bf-4a8f-4dfb-8882-98a467acb422","owner":[],"postedDate":"December 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-25T15:06:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-30 09:56:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8205131","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8205131","identity":"rs-8205131","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}