Life Cycle and Larval Feeding Habits of Macrostemum Indistinctum Banks 1911 (Trichoptera: Hydropsychidae) in the Stream Flows Into Krasiow Reservoir, Thailand

32 Journal of Food Health and Bioenvironmental Science (September - December 2020), 13(3): 32-39

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Life Cycle and Larval Feeding Habits of Macrostemum indistinctum Banks 1911 (Trichoptera: Hydropsychidae) in the Stream Flows into Krasiow Reservoir, Thailand

Orawan Khamrak & Taeng-On Prommi*

Department of Biological Science, Faculty of Liberal Arts and Science, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand

A r t i c l e i n f o A b s t r a c t

Article history: Received : 1 November 2020 The aim of this work was to determine the feeding habits of Macrostemum Revised : 27 November 2020 indistinctum larvae using gut content analysis. The study was carried out in the Accepted : 30 November 2020 stream flows into the Krasiow dam. A total of 126 larvae were captured and had the Keywords: head capsule width measured. The gut content was analyzed under microscope. Hydropsychidae, Macrostemum Seven categories of food items were determined: arthropod fragment, blue green indistinctum, Gut content, Feeding, algae, diatoms, gastropods, green algae, protozoa and rotifers. When comparing Larval stage both populations in two seasons, statistically significant differences (P<0.05) were found for gastropods and protozoa. The food items, arthropods, blue green algae, diatoms, green algae and rotifers showed no difference between both populations. Macrostemum indistinctum was found to be an omnivorous collector organism. The similarities in the seasonal trophic structure might indicate the constant availability of food.

Introduction of sediment organic matter, oxygen concentration as well as environmental conditions surrounding the watercourse Aquatic insects are one of the most dominant (Ward et al., 1995; Buss et al., 2004). They reflect elements in freshwater ecosystem trophic webs, environmental changes, aquatic insects are often used as participating in energy flow and nutrient cycling (Whiles indicators of the effects of human activity on water & Wallace, 1997). They are also important food systems, providing information on habitat and water resources for fish (Wallace & Webster, 1996) and some quality (Woodcock & Huryn, 2007). Amongst the insectivorous birds (Ward et al., 1995). The distribution aquatic insects, order Trichoptera (or caddisflies) are the and abundance of insects in freshwater systems is the most widely distributed; their larvae are common in result of complex interactions between their ecological running water (Ward, 1992) and they are one of the roles and the physicochemical conditions that relatively well-studied orders of aquatic insects in South characterize the habitat, and food availability (Merritt & East Asia (Malicky, 2010; Morse, 2017). The larvae of Cummins, 1996). Thus, the community structure depends many species coexist in running waters and are known on a number of factors, such as water quality, type of to have specific habitat and environmental requirements substrate, particle size of sediment, water flow, available (de Moor, 2007).

Life Cycle and Larval Feeding Habits of Macrostemum indistinctum Banks 1911 * Corresponding Author (Trichoptera: Hydropsychidae) in the Stream Flows into Krasiow Reservoir, Thailand e-mail: [email protected] Journal of Food Health and Bioenvironmental Science (September - December 2020), 13(3): 32-39 33

The filter-feeding caddisflies of Hydropsychidae Water samples from each collecting period were stored comprise one of the largest families of Trichoptera, with in polyethylene bottles (500 mL). Ammonia nitrogen - 2- -- about 1,756 described adult species worldwide (Morse, (NH3 N), sulfate (SO4 ), nitrate-nitrogen (NO3 N), 3- 2011). Larvae of hydropsychids live in running waters orthophosphate (PO4 ), and turbidity were determined and are generally collectors-filterers. Hydropsychidae in accordance with standard procedures (American larvae use nets spun with their silk glands to capture Public Health Association (APHA), 1992). drifting food materials in stream (Merritt & Cummins, Aquatic insects were collected during the wet season 1996). They usually construct a silken filter net at the (July to October 2019) and cold-dry season (November entrance to their fixed tubular retreat (Wiggins, 1996). to April 2019), along with 50 m stretch of the stream. Larvae present a high ecological diversity and display a Samplings were collected with a D-frame kick net with 250 wide range of tolerance to different levels of pollution, μm mesh size, including by hand picking. The caddisflies which makes them very useful organisms in biological larvae, M. indistinctunm (Fig. 2) were taken together water quality monitoring programs (Resh, 1995). The with other aquatic insects and preserved in 70% ethanol. species Macrostemum indistinctum Banks 1911 from the All the organisms were sorted and kept its in 70% Thai basin constructs a tube of sand grains (Fig. 2) on ethanol. For the M. indistinctum, head capsule width was 2 the sandy bottom of streams and utilizes an extremely use to classified into the larval instar, following the fine meshed net that captures food. However, it is not method described by MacKay (1978). Head capsule known on the trophic behavior of this species in the study 2.width Sampling was and laboratory measured analyses using an ocular micrometer. The Nine water physicochemical variables were measured once a month during January to December area during a year sampled, the aim of the present paper 2019numbers. The physicochemical of larval water instars quality parameters were weredetermined recorded directly by at the analyzing sampling site and included was to determine the feeding habits of M. indistinctum pHthe (measured frequency by a pH-meter distributions Waterproof Model Testr30of head), water capsuletemperature ( measuredwidths, by a hand-held thermometer) , and dissolved oxygen ( DO, measured by a HACH® Model sensION 6 DO meter) , total larval stages using gut analysis from the two seasons dissolvedfollowing solid ( TDS which) and electrical the conductivitywidth (rangesEC) (measured for by instarsa EURECH CyberScanwere CON110 conductivity/TDS meter). Water samples from each collecting period were stored in polyethylene bottles 2- 3- collected in corresponding on water quality measurement. (determined500 mL). Ammonia for nitrogen each (NH 3month.-N), sulfate (SO4 ), nitrate-nitrogen (NO3-N), orthophosphate (PO4 ), and turbidity were determined in accordance with standard procedures (American Public Health Association (APHAFor) 1992 a). qualitative assessment of the chief food item Aquatic insects were collected during the wet season (July to October 2019) and cold-dry season Materials and methods (ofNovember M. indistinctum, to April 2019), along witha total50 m stretch of of50 the larvaestream. Samplings of the were cold-dry collected with a D-frame kick net with 250 μm mesh size, including by hand picking. The caddisflies larvae, M. indistinctunm (Fig. 2) wereseason taken together and with66 otherlarvae aquatic ofinsects the and wetpreserved season in 70% ethanol were. dissected 1. Study area underA lla the stereomicroscope organisms were sorted and kept (Olympus its in 70% ethanol SZ51).. For the MThe. indistinctum whole, head capsule width was use to classified into the larval instar, following the method described by MacKay (1978). Head The study was conducted in the stream flows into the capsuledigestive width w astracts measured were using an removed ocular micrometer to a. Theglass number slides of larval with instars water, were determined by analyzing the frequency distributions of head capsule widths, following which the width ranges for instars Kasiow reservoir, Dan Chang district, Suphan Buri wereshredded, determined forand each examined month. under dissecting and compound For a qualitative assessment of the chief food item of M. indistinctum, a total of 50 larvae of the Province, Thailand (14°56.859´N, 099°38.118´E) (Fig. coldmicroscopes-dry season and 66 larvae(Olympus of the wet season CX31). were dissected Gut under acontents stereomicroscope were (Olympus SZ51). The whole digestive tracts were removed to a glass slide with water, shredded, and examined under dissecting 1). This stream is part of the Tha Chin River, and is 140 andseparated compound microsco intopes arthropod (Olympus CX31 ).fragment, Gut contents were blue separated green into arthropod algae, fragment, blue km long. The stream watershed is located between Khao greendiatoms, algae, diatoms, gastropods, gastropods, green green algae, protozoa algae,, and protozoa, rotifers. and rotifers.

La and Khao Yai, north of Ban Rai district, Uthai Thani Province. The study site is located at an elevation of 81 m and the stream is on average 2.0 m wide and 0.6 m deep. It presented clear water, gravel, a sandy muddy bottom, and marginal herbaceous vegetation. The collecting sites are located in the extensive areas with the sugar cane cultivation. 2. Sampling and laboratory analyses Nine water physicochemical variables were measured once a month during January to December 2019. The physicochemical water quality parameters were recorded directly at the sampling site and included pH (measured by a pH-meter Waterproof Model Testr30), water temperature (measured by a hand-held thermometer), and dissolved oxygen (DO, measured by a HACH® Model sensION 6 DO meter), total dissolved solid (TDS) and electrical conductivity (EC) (measured by a

EURECH CyberScan CON110 conductivity/TDS meter). Fig. 1 Map of the Kasiow reservoir showing the sampling site. A) Dam inlet, B) the collecting sites, and C) Fig. 1 Map Map ofof the the Kasiow Kasiow reservoir reservoir showing the sampling site (A) Dam inlet, (B) the collecting sites, and (C) Map of the Kasiow reservoir

Khamrak & Prommi Life Cycle and Larval Feeding Habits of Macrostemum indistinctum Banks 1911 (Trichoptera: Hydropsychidae) in the Stream Flows into Krasiow Reservoir, Thailand 34 Journal of Food Health and Bioenvironmental Science3 (September - December 2020), 13(3): 32-39

varied significantly during the sampling periods (p<0.05), using ANOVA (Table 1). Water temperature greatly varied from 22.37± 0.21 to 39.50±1.15°C. Temperature is one of the most important physical parameter in the biosphere, partly because it affects the movement of saturation constants of dissolved gasses in water, metabolic rate of organisms and other factors that directly or indirectly affect life on

earth (Hauer & Hill, 1996). In tropical streams, the mean Fig. 2 The characteristics of larva with case (A) and larva of Macrostemum indistinctum (B) annual temperature generally exceed 20°C (Dudgeon, 3. DataFig. analysis 2 The characteristics of larva with case (A) and larva of Macrostemum The SPSS v. 13.0 (http://www.spss.com/) was used to perform the statistical analyses. Mean, 1999). This average temperature is similar to the average standard deviation,indistinctum maximum (B) and minimum values of prey ( as the maximum and minimum number of individuals identified) were calculated for the percentage of each item of gut content. The ontogenetic shift result in this study. on diet was assessed by means of a correlation (Gamma correlation test) between larval size (measured as head3. width Data) and percentage analysis of the different items in the larval gut. To compare the food items in gut contents The mean value of pH ranged from 7.47±0.15 to between populations in cold dry and wet season, Mann-Whitney U-test was used. The SPSS v. 13.0 (http://www.spss.com/) was used 9.30±0.44. The pH is parameter concerned with the Results and discussion 1. Physicochemicalto perform parameters the ofstatistical water quality analyses. Mean, standard concentration of carbon fractions and hydrogen ions Mean values of selected physicochemical parameters of water quality at the stream flows into Kasiowdeviation, dam during this maximum study are presented and in Table minimum 1. All water parameters values such of as w preyater temperature, (as (Goldman & Horne, 1983), which shows a similarly trend dissolved oxygen (DO), pH, total dissolved solids (TDS), electrical conductivity (EC), nitrate-nitrogen (NO3- the maximum and minimum2- number of individuals of local and seasonal difference to the previous ions N) , ammonia- nitrogen ( NH3- N) , orthophosphate ( PO4 ) and turbidity varied significantly during the samplingidentified) periods (p<0.05 ), wereusing ANOVA calculated (Table 1). for the percentage of each mentioned (i.e. EC and TDS). However the result of pH Tableitem 1 Physicochemical of gut parameterscontent. of the The sampling ontogenetic site during January toshift December on 2019 diet* was Parameter/ showed significant differences. Jan Feb Mar Apr Jul Aug Sep Oct Nov Dec month Waterassessed 27.67± by30.13± means 31.30± of39 .a50± correlation29.13± 29.87±0 (Gamma29.10± 31.60± correlation 22.37± 26.80± The mean value of dissolved oxygen ranged from temp. (oC) 0.31bc 1.18de 0.53e 1.15f 0.06cd .06de 0.53cd 1.08e 0.21a 0.17b DOtest) 8.between21± 7.21± larval8.63± 6. 88±size 6. 13±(measured 6.65± 7.07± as 6.head89± 6 .99±width) 7.06± 6.13±0.16 to 8.63±0.50 mg/L. Beside the ions concentration, (mg/L) 0.13c 0.13b 0.50c 0.65ab 0.16a 0.07ab 0.03b 0.07ab 0.02b 0.04c 8.20± 7.97± 8.53± 7.87± 8.03± 8.17± 7.47± 8.30± 9.30± 8.43± pH and percentage0.10bc 0.40abc 0of.06c the0.12 abdifferent 0.06abc 0 .06itemsbc 0.15 ain the0.00bc larval0.44d gut.0.06bc dissolved oxygen is the most widely studied chemical in TDS 228.67± 111.67± 134.10± 95.93± 136.70± 162.57± 175.00± 245.67± 385.50± 359.00± (mgTo/L) compare0.58f 11.50b the0.75 cfood 1.05 a items3.91c in0. 41gutd 0.contents26e 2.52g between0.35i 1.00h aquatic environment. Dissolved oxygen greatly affects EC 457.00± 216.00± 283.33± 202.73± 289.00± 372.67± 393.33± 569.33± 602.33± 762.33± (uSpopulations/cm) 1.00g 2.00 bin cold1.53c dry0.59a and0.00 dwet 1 .53season,e 1.53f Mann-Whitney1.53h 0.58i 4.04j aquatic life as well as biochemical processes. Nearly all NH3-N 0.08± 0.69± 0.23± 0.72± 0.22± 0.20± 0.48± 0.26± 0.15± 0.24± (mg/L) 0.04a 0.34c 0.01ab 0.04c 0.13ab 0.03ab 0.30bc 0.01ab 0.03a 0.02ab NOU-3-N test4.13± was 7 .20±used. 4.10± 1.77± 1.57± 5.63± 2.93± 3.70± 4.40± NA stream organisms are sensitive to DO. Organic pollution (mg/L) 0.06bcd 0.17d 0.17bcd 0.15abc 0.46ab 4.21cd 0.06abc 0.00abcd 0.62bcd 3- PO4 1.18± 2.25± 0.84± 0.76± 0.26± 0.17± 0.40± 0.67± 1.82± 2.43± may significantly reduce DO concentration in entire (mg/L) 0.08d 0.15ef 0.04cd 0.25bcd 0.27ab 0.07a 0.19abc 0.10abcd 0.39e 0.06f Turbidity 7.39± 10.55± 4.96± 1.87± 127.33± 41.63±4 364.33± 7.39± 10.55±3 4.96± (NTUResults) 1.72a and3.36a discussion2.96a 0.75a 13.65c .97b 16.50d 1.72a .36a 2.96a stream reaches as microbial processes consume the Remarks: *The heavy rains occur in the month of May and June, environmental variables were no measured oxygen from the water (Hauer & Hill, 1996). The fact Water temperature greatly varied from 22.37± 0.21 to 39.50±1.15°C. Temperature is one of the most important1. Physicochemical physical parameter in the biosphere, parameters partly because it affects of thewater movement quality of saturation constants that DO saturation mixed well at above 80% at the of dissolved gasses in water, metabolic rate of organisms and other factors that directly or indirectly affect life on earthMean (Hauer values& Hill, 1996 of). In selected tropical streams, physicochemical the mean annual temperature generallyparameters exceed 20°C unpolluted stream, which is greatly replenished from (Dudgeon,of water 1999). This quality average temperature at the is similarstream to the averageflows result intoin this study Kasiow. dam the air, supports that DO concentrations of each during this study are presented in Table 1. All water month showed significant differences with excessive

parameters such as water temperature, dissolved oxygen concentration. (DO), pH, total dissolved solids (TDS), electrical The mean electrical conductivity values ranged from -- conductivity (EC), nitrate-nitrogen (NO3 N), ammonia- 202.73±0.59 to 602.33±0.58 μS/cm. Within the water - 2- nitrogen (NH3 N), orthophosphate (PO4 ) and turbidity column, conductivity, which indicates the ability of

Table 1 Physicochemical parameters of the sampling site during January to December 2019*

Parameter/month Jan Feb Mar Apr Jul Aug Sep Oct Nov Dec Water temp. (oC) 27.67±0.3bc 30.13±1.18de 31.30±0.53e 39.50±1.15f 29.13±0.06cd 29.87±0.06de 29.10±0.53cd 31.60±1.08e 22.37±0.21a 26.80±0.17b DO (mg/L) 8.21±0.13c 7.21±0.13b 8.63±0.50c 6.88±0.65ab 6.13±0.16a 6.65±0.07ab 7.07±0.03b 6.89±0.07ab 6.99±0.02b 7.06±0.04c pH 8.20±0.10bc 7.97±0.40abc 8.53±0.06c 7.87±0.12ab 8.03±0.06abc 8.17±0.06bc 7.47±0.15a 8.30±0.00bc 9.30±0.44d 8.43±0.06bc TDS (mg/L) 228.67±0.58f 111.67±11.50b 134.10±0.75c 95.93±1.05a 136.70±3.91c 162.57±0.41d 175.00±0.26e 245.67±2.52g 385.50±0.35i 359.00±1.00h EC (uS/cm) 457.00±1.00g 216.00±2.00b 283.33±1.53c 202.73±0.59a 289.00±0.00d 372.67±1.53e 393.33±1.53f 569.33±1.53h 602.33±0.58i 762.33±4.04j a c ab c ab ab bc ab a ab NH3-N (mg/L) 0.08± 0.04 0.69±0.34 0.23±0.01 0.72±0.04 0.22±0.13 0.20±0.03 0.48±0.30 0.26±0.01 0.15±0.03 0.24±0.02 bcd d bcd abc ab cd abc abcd bcd NO3-N (mg/L) 4.13±0.06 7.20±0.17 4.10±0.17 NA 1.77±0.15 1.57±0.46 5.63±4.21 2.93±0.06 3.70±0.00 4.40±0.62 3- d ef cd bcd ab a abc abcd e f PO4 (mg/L) 1.18±0.08 2.25±0.15 0.84±0.04 0.76±0.25 0.26±0.27 0.17±0.07 0.40±0.19 0.67±0.10 1.82±0.39 2.43±0.06 Turbidity (NTU) 7.39±1.72a 10.55±3.36a 4.96±2.96a 1.87±0.75a 127.33±13.65c 41.63±4.97b 364.33±16.50d 7.39±1.72a 10.55±3.36a 4.96±2.96a

Remarks:Remarks: *The*The heavy heavy rains rains occur occur in the in month the month of May of and May June, and environmental June, environmental variables werevariables no measured were no measured. Values with different letters indicate significant mean Valuesdifference with following different lettersTurkey indicate post hoc significant tests (P<0.05). mean difference following Turkey post hoc tests (P<0.05)

Life Cycle and Larval Feeding Habits of Macrostemum indistinctum Banks 1911 Khamrak & Prommi (Trichoptera: Hydropsychidae) in the Stream Flows into Krasiow Reservoir, Thailand Journal of Food Health and Bioenvironmental Science (September - December 2020), 13(3): 32-39 35

3- solution to carry on an electric current, is related to the PO4 concentrations varied from 0.08± 0.04 to 0.72±0.04 water fertility (Mustow, 1997). Conductivity was found mg/L, 1.57±0.46 to 7.20±0.17 mg/L, and 0.17±0.07 to as the gradual built-up along a downstream progression, 2.43±0.06 mg/L, respectively. Nitrates are the most which was clearly shown in high values in stream flow oxidized forms of nitrogen and the end product of in to the Kasiow reservoir. This may be explained by the aerobic decomposition of organic nitrogenous matter dissolution of rocks and soil nearby stream, which was (Qadri et al., 2020). Nitrogen is always present in high up at the lower altitude. Conductivity, moreover, aquatic ecosystems and is as abundant as gas in the showed evidence of seasonal pattern, which indicated a atmosphere. Relatively small quantities of nitrogen exist high value in hot-dry season and less value in cold-dry in the combined forms of ammonia, nitrate, nitrite, urea, season and wet season, respectively. This pattern was and dissolved organic compounds. Nitrate is usually explained by the theory of Bishop (1973). In hot-dry the most important nutrient. Natural changes in the season, the amount of the falling leaves or leaf litters were vegetation of the drainage basin caused by fire, floods, high. Leaves were quickly decomposed by high temperature or artificial clearing usually results in an increase of and then, the soluble nutrients were released into the water. nitrate in streams. Even moderate environmental The mean total dissolved solids values ranged from disturbances, such as sensible farming or logging without 95.93±1.05 mg/L to 385.50±0.35 mg/L. Total dissolved severe erosion, release a higher quantity of nitrate more solids is another parameter, which indicates the materials than ammonia or phosphate (Goldman & Horne, 1983). that are chemically dissolved in water. This includes Therefore nitrate concentration also reflects the range of materials such as calcium, chloride, sodium, magnesium, pollution or disturbances, while ammonium concentration silicate and carbonate. The TDS enters the stream from indicates a metabolic waste product of animals. At the three natural sources; (1) atmosphere (i.e. rainfall), time of this study nitrate concentrations were found (2) soil and rock weathering and (3) biological process to be slightly low. Although it should be noted that (Webster & Ehrman, 1996). Seasonal pattern of TDS according to Goldman & Horne (1983) indicated that indicates that a high concentration of TDS appears the concentration of most nitrogen compounds in lake in hot-dry season and is less concentration in cold- and stream tended to follow regular seasonal pattern. The dry season. This shows a similar trend to electrical extreme values in this study in wet and hot-dry season, conductivity parameter and can be explained by the same respectively, coincide with the recorded heavy rain before explanation. collecting began the same as the value of turbidity and The mean turbidity values ranged from 1.87±0.75 to others. The result of ammonia was also similar to nitrate 364.33±16.50 NTU. The high turbidity was recorded concentration. Phosphate, in contrast to nitrate, is during the wet season due to heavy rainfall. Turbidity readily adsorbed to soil particles and does not move of the water is caused by suspended solids and any easily in groundwater. High flows of total phosphorus is coloration produced by dissolved substance. The source due to erosion of particles from steep slopes with easily of suspended solids and dissolved substance can be erodible soils. Agricultural, domestic and industrial inorganic particles and organic debris from soil erosion wastes are the major sources of soluble phosphate. in the agricultural areas, with high erosion during the Phosphate containing detergents, for example, commonly flood (Bisson & Montgomery, 1996). As results of this contribute about half the phosphorus contained in study indicated the turbidity of all month were different, domestic sewage. Here the detected phosphorus is especially in the month of rainy season. The adverse treated as the reflection of waste within water column effects of turbidity on freshwater system included; low and hill-slope. The amounts of phosphorus concentration penetration of light which then reduces primary and has significant seasonal pattern similarly to the results secondary production, high adsorption of nutrient of the nitrate and ammonia. molecules to suspended materials making the nutrients 2. Larval instars of Macrostemum indistinctum unavailable for plankton production, decreased oxygen From the specimens collected on all occasions during concentration, and clogged filter-feeding apparatus, and the wet season (July to October 2019) and cold-dry digestive organs of planktonic organisms, which may season (November to April 2019), a total of 5,583 adversely affect the production of larvae (Gupta & individual of hydropsychid larvae were found. 126 larvae Gupta, 2006). of M. indistinctum were identified and measured of - -- The mean dissolved nutrients, NH3 N, NO3 N, and head capsule width.

Khamrak & Prommi Life Cycle and Larval Feeding Habits of Macrostemum indistinctum Banks 1911 (Trichoptera: Hydropsychidae) in the Stream Flows into Krasiow Reservoir, Thailand 36 Journal of Food Health and Bioenvironmental Science (September - December5 2020), 13(3): 32-39

Larval head width ranged from 0.13 to 1.98 mm, which by M. indistinctum larvae were diatoms, green algae and as the reflection of wasteall developmentalwithin water column stages and hillof -larvaeslope. The were amounts present of phosphorus in our concentrationblue green algaehas (Tables 3-4 and Fig. 4). significant seasonal patternanalysis similarly (Table to the2). resultsThe larvae of the nitrate were andclassified ammonia .into five In the cold-dry season, 50 larvae were analyzed gut instars using 126 specimens. Head widths of first instars content. The most abundant trophic resource was diatoms 2. Larval instars of Macrostemumranged from indistinctum 0.13-0.18 mm (n = 8). Head widths of followed by green algae, blue green algae, gastropods, From the specimens collected on all occasions during the wet season (July to October 2019) and cold-dry season (Novembersecond toinstars April 2019 ranged), a totalfrom of 0.265,583 toindividual 0.44 mm of hydropsychid (n = 18). larvaearthropods, were found rotifers. and protozoa (Table 3 and Fig. 4). 126 larvae of M. indistinctumThe third were instars identified ranged and measuredfrom 0.50 of tohead 0.81 capsule mm width(n = .30). Significant positive correlations (P<0.05) was found Larval head Thewidth fourth ranged instars from 0 ranged.13 to 1 .from98 mm, 0.83 which to 1.36 all developmental mm (n = 34) stages between of larvae headwere width and percentage of arthropods present in our analysisand (Table the 2fifth). The instars larvae were ranged classified from into 1.38 five instarsto 1.98 using mm 1 2 6 specimens(Gamma. correlationHead = 0.707), blue green algae (Gamma widths of first instars ranged from 0.13-0.18 mm (n = 8). Head widths of second instars ranged from 0.26 to 0.44 mm (n = 18). The(n =third 34) instars (Fig. ranged 3 and from Table 0. 502). to 0.81 mm (n = 30). The fourth instarscorrelation ranged from = 0.352), diatoms (Gamma correlation = 0.83 to 1.36 mm (n = 34) and the fifth instars ranged from 1.38 to 1.98 mm (n = 34) (Fig. 3;0.587), Table 2 ).green algae (Gamma correlation = 0.563), and rotifers (Gamma correlation = 1.000). No significant positive correlations was found between larval stages and percentage of gastropods (Gamma correlation = –0.034) and protozoa (Gamma correlation = –1.000). The example of food items in the gut of M. indistinctum was shown in Fig. 5.

Table 3 Gut contents of the larvae of Macrostemum indistinctum* in the dry season

Food item/ Instar II Instar III Instar IV Instar V individual N Mean N Mean N Mean N Mean Arthropods 6 1.2±0.4 15 1.5± 0.9 14 3.9±6.8 15 5.6±3.3 (1-2) (1-4) (1-24) (1-13) Blue green 6 9.6±13.2 15 9.2± 8.5 14 11.6±10.2 15 32.6±45.2 algae (1-39) (1-33) (1-34) (1-176) Diatoms 6 45.3±65.2 15 64.5±11 14 165.9±113.1 15 218.5±142.9 (9-211) (11-155) (13-415) (26-573) Gastropods 6 15.7±16.0 15 9.1± 8.6 14 9.2±6.1 15 8.5±6.7 (1-34) (2-29) (2-20) (3-20) Green algae 6 6.7±6.5 15 10.0±5.2 14 20.4±9.8 15 37.4±25.2 (1-35) (1-17) (3-32) (1-74) Protozoas NA NA 15 1.3± 0.6 14 1.0±00 15 1.0±00 (1-2) (1) (1) Rotifers NA NA 15 1.0± 0.0 14 1.0±00 15 13.6±14.0 (1) (1) (3-38) Fig. 3 The frequencyFig. distribution 3 The frequency of larval distribution instars ofof larval M. indistinctuminstars of M. indistinctumat all sampling at all sites, based on head Remarks: *Gut content of first instar was not dissected for qualitative assessment capsule width sampling sites, based on head capsule width

Table 2 Median and Tablerange 2 ofMedian head and capsule range widthof head ( capsulemm) forwidth larval (mm) instars for larval of Minstars. indistinctum of Induring the wet study season, 66 larval analyzed in this study had period M. indistinctum during study period gut content, and they ingested mainly diatoms followed Larval instar NumberLarval measured Number MeanMean head head width width (mm )Range of headRange width of head widthby green (mm algae,) blue green algae, gastropods, arthropods, I 8 instar measured 0.16±0.02(mm) (mm)0.13-0.18 rotifers and protozoa (Table 4 and Fig. 4). A significant II 18 I 8 0.34±0.050.16±0.02 0.13-0.180.26-0.44 positive correlation (P<0.05) was found between head III 30 II 18 0.62±0.070.34±0.05 0.26-0.440.50-0.81 IV 34 III 30 1.06±0.080.62±0.07 0.50-0.810.83-1.36 width and percentage of arthropods (Gamma correlation V 34 IV 34 1.63±0.091.06±0.08 0.83-1.361.38-1.98 =0.635), blue green algae (Gamma correlation = 0.660), V 34 1.63±0.09 1.38-1.98 diatoms (Gamma correlation = 0.594), green algae 3. Larval gut contents in Macrostemum indistinctum (Gamma correlation = 0.589), and protozoa (Gamma Larval gut contents were assessed qualitatively. Gut contents were separated into arthropod fragments, blue green3. algae, Larval diatoms, gut contentsgastropods, in green Macrostemum algae, protozoa indistinctum and rotifers. Gut correlationcontent analysis = 0.280). No significant correlations was indicated that larvae are omnivorousLarval gut filterers contents. The were major assessed resource itemsqualitatively. used by M .Gut indistinctum found larvae between were head width and percentage of gastropods diatoms, green algae contentsand blue green were algae separated (Tables into3-4, Fig arthropod. 4). fragments, blue (Gamma correlation = 0.103) and rotifers (Gamma In the cold-drygreen season, algae, 50 diatoms,larvae were gastropods, analyzed gut green content algae,. The mostprotozoa abundant correlationtrophic resource = –0.600). was diatoms followed by green algae, blue green algae, gastropods, arthropods, rotifers and protozoa (Table 3, Fig. 4). Significantand positive rotifers. correlations Gut content (P<0 .analysis05) was foundindicated between that head larvae width and percentageComparing of populations in two seasons, statistically are omnivorous filterers. The major resource items used significant differences (P<0.05) were found for

(Mann-Whitney U = 0.043). The food items, arthropods 0.7%. Chaetas of oligochaeta, eggs, legs and tegument (Mann–Whitney U test, P = 0.564), blue green algae of mayflies and antennas, among others, were observed The feeding habits of M. indistinctum populations in the stream flows into Kasiow reservoir widely (Mann–Whitney U test, P = 0.773), diatoms (Mann– coincidein dthe with thatinvertebrate observed in other speciesremains. of Hydropsychidae larvae populations in Thailand (Maneechan et al., 2018; Thamsenanupap & Prommi, 2020). They are mainly filtering-collectors, but detritus is also an Whitney U test, P = 1.000), green algae (Mann–Whitney important Hydropsychidaecomponent of their smaller larvae larvae diet. Among are the food described items, diatoms, greenas algaefilterer and blue green algae are also the major resources for most of the studied Hydropsychidae species (Maneechan et al., 2018; U test, P = 0.386) and rotifers (Mann–Whitney U test, Thamsenanupapcollectors & Prommi, (Merritt 2020). When & analyzing Cummins, changes in diet1984). with growth, A apartfilterer from a gen eral decrease in detritus intake, they seemed to be a significant trend to ingest higher size prey (as gastropods, P = 0.796) showed no difference between both populations. protozoacollector and rotifers is) by classified bigger larvae. This by was the the resultway of thatdifferent they conditions eat: and thoseenvironmental parameterswhich at the feed sampling on site seston (Table 1 ).moved The same resultby awas current, published by using Gil et al .silk (2008 ).nets During both periods the most representative items were invertebrates and amorphous substance constituting about 70% of Table 4 Gut contents of the larvae of Macrostemum indistinctum* in the wet the dietor. Hydropsychidae,body parts Smicridea (passive), (Rhyacophylax and) dithyrathose Flint, which 1974 between resuspension both periods preferred season amorphous material (37.4%), invertebrate remains (32.6%), filamentous algae (11.5%), leaves fragments (6.3%)deposits, inorganic matter which (5.9%) are and unicellular filtered algae using(4.8%). Hyphae,silk finenets sediment or body and pollen parts were found Food item/ Instar II Instar III Instar IV Instar V in small(active) percentages Palmer between 0 and& 0O´Keefe.7%. Chaetas of (1992).oligochaeta, eggs,Gallardo-Mayenco legs and tegument of mayflies and individual antennas, among others, were observed in the invertebrate remains. N Mean N Mean N Mean N Mean Hydropsychidae larvae are described as filterer collectors (Merritt & Cummins, 1984). A filterer collectoret isal. classified (1998) by the way that described they eat: those which the feed on feeding seston moved by habits a current, using of silk nets Arthropods 12 1.0±0.0 15 1.4±0.5 20 1.6±1.3 19 4.4±4.1 or bodyHydropsychidae parts (passive), and those which in resusprelationension deposits to the which different are filtered using net silk nets sizes or body parts (1) (1-2) (1-6) (1-15) ( active) Palmer & O´Keefe ( 1992). Gallardo- Mayenco et al. (1998) described the feeding habits of Blue green 12 3.2±1.3 15 8.8±7.7 20 20.5±28.5 19 43.8±19.7 Hydropsychwhichidae canin relation be torelated the different to net the sizes whichlarvae can be development related to the larvae development stage. stage. algae (1-4) (1-27) (2-119) (10-77) Diatoms 12 11.4 ±10.8 15 86.6 ±65.3 20 126.1±115.8 19 269.2±166.0 Arthropods Blue green algae Diatoms Gastropods (1-38) (1-223) (1-386) (7-659) Gastropods 12 4.2± 3.2 15 6.7 ±6.1 20 3.0±2.9 19 8.1±6.3 (1-10) (1-20) (1-11) (1-24) Instar V Green algae 12 2.7± 2.2 15 28.9±25.1 20 52.8±68.9 19 113.3±102.6 (1-7) (2-82) (2-254) (4-363) Instar IV

Protozoa NA NA 15 2.0±1.4 20 2.0±1.7 19 3.2±2.8 Instar III (1-3) (1-4) (1-8) Wet season Rotifers NA NA 15 1.0±00 20 2.0±00 19 1.0±00 Instar II (1) (2) (1) Instar V Remarks: *Gut content of first instar was not dissected for qualitative assessment Instar IV The feeding habits of M. indistinctum populations in Instar III the stream flows into Kasiow reservoir widely coincided Dry season with that observed in other species of Hydropsychidae Instar II larvae populations in Thailand (Maneechan et al., 2018; 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Frequency (number of organisms with content) Thamsenanupap & Prommi, 2020). They are mainly Fig. 4 Frequency of food items in the gut of Macrostemum indistinctum filtering-collectors, but detritus is also an important Fig. 4 Frequency of food items in the gut of Macrostemum indistinctum component of their smaller larvae diet. Among the The food item composition in larval gut observed in M. indistinctum obtained from the stream flows into the Kasiow reservoir was in agreement with the genus description due to the variety of ingested food food items, diatoms, green algae and blue green algae items. ConsideringThe food the size item of the items composition found in the digestive in tractslarval of M . gutindistinctum observed larvae, the in definition given for omnivorous collectors might be extended since they have been traditionally described as processors are also the major resources for most of the studied of fineM. particulat indistinctume organic matter (obtainedFPOM) and coarse from particulate the organic stream matter (flowsCPOM). However, into some cases, coarse particulate organic matter (CPOM), such as complete preys or fragments of big filamentous Hydropsychidae species (Maneechan et al., 2018; algae,the were Kasiowfound. reservoir was in agreement with the genus descriptionThe similarities founddue in tothe seasonalthe variety trophic struc tureof mightingested indicate the food constant items.availability of the Thamsenanupap & Prommi, 2020). When analyzing food resource. This possibility was reinforced by some studies carried out with coleopterans and dipterans, changes in diet with growth, apart from a general decrease Considering the size of the items found in the digestive in detritus intake, they seemed to be a significant trend tracts of M. indistinctum larvae, the definition given for to ingest higher size prey (as gastropods, protozoa and omnivorous collectors might be extended since they rotifers) by bigger larvae. This was the result of different have been traditionally described as processors of fine conditions and environmental parameters at the sampling particulate organic matter (FPOM) and coarse particulate site (Table 1). The same result was published by Gil et organic matter (CPOM). However, some cases, coarse al. (2008). During both periods the most representative particulate organic matter (CPOM), such as complete items were invertebrates and amorphous substance preys or fragments of big filamentous algae, were found. constituting about 70% of the diet. Hydropsychidae, The similarities found in the seasonal trophic structure Smicridea (Rhyacophylax) dithyra Flint, 1974 between might indicate the constant availability of the food both periods preferred amorphous material (37.4%), resource. This possibility was reinforced by some studies invertebrate remains (32.6%), filamentous algae (11.5%), carried out with coleopterans and dipterans, which leaves fragments (6.3%), inorganic matter (5.9%) and showed that a change in the trophic group involved a unicellular algae (4.8%). Hyphae, fine sediment and change in the proportion of items consumed during the dry and wet seasons (Motta & Uieda, 2004).

Khamrak & Prommi Life Cycle and Larval Feeding Habits of Macrostemum indistinctum Banks 1911 (Trichoptera: Hydropsychidae) in the Stream Flows into Krasiow Reservoir, Thailand 8

38which showed that aJournal change of Food in Health the trophicand Bioenvironmental group involved Science (September a change - December in 2020),the proportion13(3): 32-39 of items consumed during the dry and wet seasons (Motta & Uieda, 2004).

Fig. 5 Food itemsFig. in 5 Foodthe itemsgut inof the M gut. indistinctumof M. indistinctum underunder bright bright field microscopefield microscope (magnification ( magnificationx40) x40)

ConclusionConclusions References Most of M. indistinctum consumed diatoms, blue green algae, and green algae as the main food source.Most The of small M. indistinctum larval of M consumed. indistinctum diatoms, consumed blue Americandetritus asPublic part Health of their Association diet, whereas (APHA). the (1992). larger Standard larval green algae, and green algae as the main food source. methods for the examination of water and wastewater consumed larger food items. M. indistinctum is not exclusively thherbivorous, but rather omnivorous species The small larval of M. indistinctum consumed detritus (18 ed.). Washington, D.C.: American Public Health with flexible feeding habits. The physico-chemical of water qualityAssociation. parameters such as electrical conductivity, astotal part dissolved of their diet, solids, whereas water the turbidity, larger larval orthophosphate, consumed Bishop,pH, water J.E. temperature, (1973). Limnology dissolved of a smalloxygen, Malayan ammonia river - largernitrogen, food and items. nitra teM.-nitrogen indistinctum are currently is not exclusively affecting on the lifeSungai stages Gombak. of M. indistinctum Canada: W. Junk, in Kasiow The Hague. reservoir, herbivorous,Thailand. Hydropsychidae but rather omnivorous are richly species diverse with flexiblein Thai streamsBisson, and P.A., other & Montgomery, species can D.R. be expected (1996).Valley to be segments, affected feedingin the same habits. way The. physico-chemical of water quality stream reaches, and channel units. In F.R. Hauer & parameters such as electrical conductivity, total dissolved G.A. Lamberti (Eds), Methods in Stream Ecology. San Diego: Academic Press. solids,Acknowledgement water turbidity, orthophosphate, pH, water Buss, D.F., Baptista, D.F., Nessimain, J.L., & Egler, M. (2004). temperature,This dissolved research oxygen, work was ammonia-nitrogen, supported by the and Faculty of LiberalSubstrate Arts specificity, and Science, environmental Kamphaeng degradation Saen and nitrate-nitrogenCampus, Kasetsart are currently University affecting. on the life stages disturbance structuring macroinvertebrate assemblages in neotropical streams. Hydrobiologia, 518(1-3), 179- of M. indistinctum in Kasiow reservoir, Thailand. Hydropsychidae are richly diverse in Thai streams and 188. References de Moor, F.C. (2007). Regional biogeographical differences in other species can be expected to be affected in the same American Public Health Association (APHA). (1992). StandardTrichoptera methods diversity for the inexamination South Africa: of waterObserved and way. th patterns and processes. In J. Bueno-Soria R. Barba- wastewater (18 ed.). Washington, D.C.: American Public Health Association. nd Bishop, J.E. (1973). Limnology of a small Malayan river SungaiAlvares Gombak & B. Armitage. Canada (Eds.),: W. Junk,Proceedings The Hague of the 12. Acknowledgement International Symposium on Trichoptera (pp. 211-218). Bisson, P.A., & Montgomery, D.R. (1996).Valley segments,USA: stream Caddis reaches, Press. and channel units. In Hauer F.R. & Lamberti G.A. (Eds), Methods in StreamDudgeon, Ecology D.. (1999).San Diego Tropical: Academic Asian streams: Press. Zoobenthos, This research work was supported by the Faculty Buss, D. F. , Baptista, D. F. , Nessimain, J. L. , & Egler, M. ecology(2004). and Substrate conservation. specificity, Hong Kong: environmental Hong Kong of Liberal Arts and Science, Kamphaeng Saen Campus, degradation and disturbance structuring macroinvertebrateUniversity assemblages Press. in neotropical streams. Kasetsart University. Gallardo-Mayenco, A., Prenda, J., & Toja, J., (1998). Spatio Hydrobiologia, 518(1-3), 179–188. - Temporal distribution and ecological preferences of de Moor, F. C. (2007). Regional biogeographical differencescoexisting in Trichoptera Hydropsychid diversity species (Trichoptera) in South Africa in two : Observed patterns and processes. In J. Bueno-SoriaMediterranean R. Barba- RiverAlvares Basins & B(S. Spain). Armitage International ( Eds. ) , nd Review of Hydrobiology, 83(2), 123–134. Proceedings of the 12 International Symposium on Trichoptera (pp. 211–218). USA: Caddis Press. Dudgeon, D. (1999). Tropical Asian streams: Zoobenthos, ecology and conservation. Hong Kong: Hong Life Cycle andKong Larval University Feeding Habits Press of Macrostemum. indistinctum Banks 1911 Khamrak & Prommi (Trichoptera:Gallardo- Mayenco,Hydropsychidae) A. in, Prenda,the Stream JFlows. , & into Toja, Krasiow J. ,Reservoir, ( 1998). Thailand Spatio - Temporal distribution and ecological preferences of coexisting Hydropsychid Species (Trichoptera) in two Mediterranean River Basins (S Spain). International Review of Hydrobiology, 83(2), 123–134. Gil, M.A., Tripole, S., & Vallania, E.A. (2008). Feeding habits of Smicridea (Rhyacophylax) dithyra Flint, 1974 (Trichoptera: Hydropsychidae) larvae in the Los Molles stream (San Luis - Argentina).

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Khamrak & Prommi Life Cycle and Larval Feeding Habits of Macrostemum indistinctum Banks 1911 (Trichoptera: Hydropsychidae) in the Stream Flows into Krasiow Reservoir, Thailand