Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Summary

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

Abstract

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water’s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Introduction

Biological monitoring programs, which use living organisms to evaluate environmental health, are often used to assess water quality or monitor success of ecosystem restoration programs. Rapid bioassessment protocols (RBP) using benthic macroinvertebrate assemblages have been popular among state water resource agencies since 1989¹. Traditional methods of sampling benthic macroinvertebrates for RBPs, such as the dip-net, Surber sampler, and Hess sampler², can be time-consuming, expensive, and may only measure assemblages from a particular microhabitat³. An efficient, alternative RBP for generating biological information about a particular water body involves collection of Chironomidae surface-floating pupal exuviae (SFPE)³.

The Chironomidae (Insecta: Diptera), commonly known as non-biting midges, are holometabolous flies that typically occur in aquatic environments before emerging as adults on the water’s surface. The chironomid family is species-rich, with approximately 5,000 species described worldwide; however, as many as 20,000 species are estimated to exist⁴. Chironomids are useful in documenting water and habitat quality in many aquatic ecosystems because of their high diversity and variable pollution tolerance levels⁵. Furthermore, they are often the most abundant and widespread benthic macroinvertebrates in aquatic systems, typically accounting for 50% or more of the species in the community^5,6. Following emergence of the terrestrial adult, the pupal exuviae (cast pupal skin) remains floating on the water’s surface (Figure 1). Pupal exuviae accumulate along banks or behind obstructions through the action of wind or water current and can be easily and rapidly collected to give a comprehensive sample of chironomid species that have emerged during the previous 24-48 hr⁷.

The relative abundance and taxonomic composition of collected SFPE reflects water quality, considering that some species are very pollution tolerant, while others are quite sensitive⁵. The SFPE method has many advantages over traditional larval chironomid sampling techniques including: (1) minimal, if any, habitat disturbance occurs at a sampling area; (2) samples do not focus on collecting living organisms, but rather the non-living skin, so the trajectory of community dynamics is not affected; (3) identification to genus, and often species, is relatively easy given appropriate keys and descriptions³; (4) collecting, processing, and identifying samples is efficient and economical in comparison to traditional sampling methods^3,8,9; (5) accumulated exuviae represent taxa that have originated from a wide range of microhabitats¹⁰; (6) the method is applicable in nearly all aquatic environments, including streams and rivers, estuaries, lakes, ponds, rock pools, and wetlands; and (7) SFPE maybe be a more sensitive indicator of ecosystem health since they represent individuals that have completed all immature stages and successfully emerged as adults¹¹.

The SFPE method is not a new approach for gathering information about chironomid communities. Use of SFPE was first suggested by Thienemann¹² in the early 1900s. A variety of studies have used SFPE for taxonomic surveys (e.g., ^13-15), biodiversity and ecological studies (e.g. ^7,16-19), and biological assessments (e.g.,^20-22). Additionally, some studies have addressed different aspects of sample design, sample size, and number of sample events required for achieving various detection levels of species or genera (e.g.,^8,9,23). These studies indicate that relatively high percentages of species or genera can be detected with moderate effort or expense associated with sample processing. For example, Anderson and Ferrington⁸ determined that based on a 100-count subsample,  1/3^rd less time was required to pick SFPE samples compared to dip-net samples. Another study determined that 3-4 SFPE samples could be sorted and identified for every dip-net sample and that SFPE samples were more efficient than dip-net samples at detecting species as species richness increased³. For example, at sites with species richness values of 15-16 species, the average dip-net efficiency was 45.7%, while SFPE samples were 97.8% efficient³.

Importantly, the SFPE method has been standardized in the European Union²⁴ (known as chironomid pupal exuviae technique (CPET)) and North America²⁵ for ecological assessment, but the method has not been described in detail. One application of the SFPE methodology was described by Ferrington, et al.³; however, the primary focus of that study was to evaluate the efficiency, efficacy, and economy of the SFPE method. The purpose of this work is to describe all steps of the SFPE method in detail, including sample collection, laboratory processing, slide mounting, and genus identification. The target audience includes graduate students, researchers, and professionals interested in expanding traditional water quality monitoring efforts into their studies.

Protocol

1. Preparation of Field Collection Supplies

1. Determine the number of SFPE samples that should be collected based on the study design and acquire one sample jar (e.g., 60 ml) for each sample.
2. Prepare two date and locality labels for each sample jar. Place one on the inside and affix the other to the outside of the jar. Ensure that each date and locality label includes the following information:  country, state, county, city, water body, GPS coordinates, date, and name of person(s) collecting the sample.
3. Gather other specific materials and equipment (see Table of Specific Materials/Equipment).

2. Field Collection

1. Hold a larval tray in one hand and a sieve in the other. Dip the larval tray into the water where SFPE accumulate (e.g., foam accumulations, snags, emergent vegetation, debris, back eddies, and along bank edges) (Figure 2A), allow water, exuviae, and debris to enter the larval tray, and pour this material through the sieve. If sampling in a lotic system, begin at the downstream end of the sample reach and work upstream (Figure 2B). If sampling in a lentic system, begin at the downwind shoreline. 
   1. Repeat step 2.1 for 10 min (or as otherwise defined for a specific sampling regime) within each pre-defined sample reach (typically 100-200 m for samples collected from streams, but dependent on the overall area of the aquatic monitoring site); move between SFPE accumulation areas as appropriate.
2. Concentrate debris in one area of the sieve using a squirt bottle filled with water from the sample site and carefully transfer SFPE sample to pre-labeled sample jar with the aid of forceps and a stream of ethanol from a squirt bottle. Fill sample jar with ethanol.
3. Repeat steps 2.1 to 2.2 for all samples.

3. Sample Picking

NOTE: The rest of this protocol pertains to a 300 SFPE subsample and may need to be modified for other subsample sizes. See Bouchard and Ferrington's⁹ subsampling and sampling frequency guidelines for tailoring SFPE methods to meet study-specific goals and resources.

1. Allocate a 1-dram vial for each SFPE sample; prepare a date and  locality label to place inside each vial and fill the vial ¾ full with ethanol.
2. Remove lid from the corresponding sample jar and check for attached pupal exuviae. Gently rinse contents off the lid onto a Petri dish using a squirt bottle filled with ethanol. Locate and remove label from the inside of the sample jar using forceps and gently rinse contents off the label onto the Petri dish. Set label aside.
3. Transfer the contents of the sample jar into a larval tray, rinsing with ethanol to ensure no SFPE remain in the sample jar. Transfer a portion of the pupal exuviae, residue, and ethanol from the tray to the Petri dish. Ensure that the sample is covered in ethanol. 
4. Place the Petri dish under a stereo microscope. Systematically scan the contents of the Petri dish for pupal exuviae. Pick all pupal exuviae from the dish using forceps and place into the vial. Do not pick specimens that are broken (i.e., do not have at least half of the cephalothorax and abdomen), dried, or compressed to avoid later identification problems.
   NOTE: Identification to species often requires that the entire specimen is present, though in some cases, genus-level identification may be possible with partial specimens.
   1. Swirl dish and scan for additional pupal exuviae, including any that could be stuck to sides of the dish, as well as, any small and translucent specimens that may not have be detected initially. Repeat until two consecutive scans reveal no additional pupal exuviae.
5. Repeat steps 3.3 and 3.4 until all or 300 pupal exuviae have been picked. When 300 pupal exuviae have been picked, return the residue from the Petri dish to the larval tray and rinse the Petri dish with ethanol. Then, transfer the residue from the larval tray to the empty sample jar, add the date and locality label, and put the lid on  the jar. Retain or dispose of residue according to project-specific protocols.

4. Sample Sorting

1. Pour all picked pupal exuviae from the labeled vial into a Petri dish filled with enough ethanol to just cover specimens.
2. Under a stereo microscope, separate specimens into distinct morphological groups (i.e., morphotaxa) and place each morphotaxon into separately a labeled vials filled 3/4^th full with ethanol.
   1. Utilize external morphological characteristics to separate chironomid morphotaxa. For example, from the cephalothorax, use differences in the presence, size, shape, and coloration of the cephalic tubercles, frontal warts, frontal setae, and thoracic horn. From the abdomen, use spines, hookrows, shagreen, setae, and spurs of the abdominal segments, in addition to the anal lobes for morphotaxa separation (Figure 4A). See Ferrington, et al. ^5,Sæther ^26,Pinder and Reiss ²⁷ for additional descriptions and figures of morphological characteristics.
   2. Use additional ethanol if specimens begin to dry.

5. Slide Mounting

1. Fill one well of a multi-well plate for each morphotaxon with 95% ethanol.
   1. Place multiple representations (e.g., 25% of total) of each morphotaxon to be slide mounted into individual wells of the plate. Allow specimens to sit in well for at least 10 min to dehydrate sufficiently.
2. Label slides with appropriate site, collection, and identification information (Figure 3).
3. Place slide on the stereo microscope.
   NOTE: A template of the slide taped to the stage is useful for consistent placement.
4. Place a drop of Euparal on the slide; spread the Euparal so that it approximates the size of the coverslip. Use proper ventilation when working with Euparal.
   NOTE: Use proper ventilation when working with Euparal.
5. Embed a representative from the first morphotaxon into the Euparal using forceps.
   NOTE: To void excess ethanol from the specimen, using a forceps, gently tap specimen on laboratory wipes  prior to embedding it in Euparal.
6. Separate the cephalothorax from the abdomen using fine-tipped forceps and/or dissection probes (Figure 4A).
   1. Split the cephalothorax along the ecdysial suture (Figure 4B) and open the cephalothorax so that the suture edges are on opposite sides (Figure 4C).
   2. Orient the cephalothorax so that the ventral side is facing up (Figure 4C).
   3. Position the abdomen dorsal side up; place immediately below the cephalothorax (Figure 4C).
7. Place a coverslip on the specimen. Hold coverslip at an angle, with one edge touching the slide, and then slowly lower and drop the coverslip to reduce air bubble formation. Press lightly on the coverslip to flatten the specimen.
8. Repeat steps 5.3 through 5.7 for all dehydrated specimens.

6. Genus Identification

1. Determine genus of slide-mounted specimens using a compound microscope. Identify specimens to genus using keys and diagnoses in Wiederholm 28 and Ferrington, et al.⁵. If needed, confirm family-level identification using Ferrington, et al.⁵.   NOTE: There have been numerous generic descriptions and revisions since Wiederholm²⁸ and Ferrington, et al.⁵, therefore, these keys and diagnoses are incomplete and need to be supplemented with primary literature.

Results

Figure 1 illustrates the chironomid life cycle; immature stages (egg, larva, pupa) typically take place in, or closely associated with, an aquatic environment. Upon completion of the larval life stage, the larva constructs a tube-like shelter and attaches itself with silken secretions to the surrounding substrate and pupation occurs. Once the developing adult has matured, the pupa frees itself and swims to the surface of the water where the adult can emerge from the pupal exuviae. The exuviae fills with ...

Discussion

The most critical steps for successful SFPE sample collection, picking, sorting, slide mounting, and identification are: (1) locating areas of high SFPE accumulation within the study area during field collection (Figure 2A); (2) slowly scanning the contents of the Petri dish for detection of all SFPE during sample picking; (3) developing the necessary manual dexterity to dissect the cephalothorax from the abdomen during slide mounting (Figure 4A); and (4) recognizing key morphologic...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ethanol	Fisher Scientific	S25309B	70-95%
Plastic wash bottles	Fisher Scientific	0340923B
Sample jar	Fisher Scientific	0333510B	Glass or plastic, 60-mL recommended
Testing sieve	Advantech	120SS12F	125-micron mesh size
Larval tray	BioQuip	5524	White
Stereo microscope
Glass shell vials	Fisher Scientific	0333926B	1-dram size
Plastic dropper	Thermo Scientific	1371110	30 to 35 drops/mL
Fine forceps	BioQuip	4524	#5
Petri dish	Carolina	741158	Glass or plastic
Multi-well plate	Thermo Scientific	144530	Glass or plastic
Glass microslides	Thermo Scientific	3010002	3 x 1 in.
Glass cover slips	Thermo Scientific	12-519-21G	Circular or square
Euparal mounting medium	BioQuip	6372B
Pigma pen	BioQuip	1154F	Black
Probe	BioQuip	4751
Kimwipes	Kimberly-Clark Professional™	34120