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Field and laboratory studies were conducted to evaluate the use of bacterial growth on aquatic insects as a metric for determining the existence of nutrient impacts in wetlands. Results from field investigations indicated that elevated concentrations of nitrate and phosphate...

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  1. WETLANDS, Vol. 20, No. 1, March 2000, pp. 91–100 © 2000, The Society of Wetland Scientists AN INSECT-BACTERIA BIOINDICATOR FOR ASSESSING DETRIMENTAL NUTRIENT ENRICHMENT IN WETLANDS 2 A. Dennis Lemly¹ and Ryan S. King ¹ United States Forest Service Southern Research Station Coldwater Fisheries Research Unit Department of Fisheries and Wildlife Sciences Virginia Tech University Blacksburg, V irginia , USA 24061-0321 2 Duke University Wetland Center Nicholas School of the Environment Durham, North Carolina, USA 27708 Abstract : Fiel d and laborator y studie s wer e conducte d to evaluat e the use of bacteria l growt h on aquatic insect s as a metri c fo r determinin g the existenc e of nutrien t impact s in wetlands . Result s fro m field inves- tigation s indicate d that elevate d concentration s of nitrat e and phosphat e wer e associate d with growt h of filamentous bacteria on insect body surfaces and that there were significantly fewer mayflies (Ephemeroptera) in th e nutrient-enriche d wetland . Laborator y investigation s confirme d a stron g linkag e betwee n bacterial growt h and reduce d surviva l of mayflies . Surviva l was examine d for individual s with bacteria l infestation rangin g from 0% to 60 % bod y coverage . A threshol d for catastrophi c mortalit y was presen t at about the 25% leve l of coverage ; ther e wer e ver y few survivor s abov e that level . Base d on thes e findings , th e diag- nosti c endpoin t fo r th e bioindicato r is 25% bod y coverag e by bacteria l growth , a leve l that signifie s major differences in insect populations in the field and is also easy to detect visually. This study provides evidence that th e insect-bacteri a bioindicato r is a reliabl e too l for assessin g nutrien t impact s on wetlan d macroinver- t ebrat e c ommunities . T h e b ioindicato r c oul d b e u sefu l i n t h e d evelopmen t o f a W etlan d Bioassessment Protocol. Ke y Words : bioindicator , nutrien t pollution, eutrophication , macroinvertebrates , aquati c bacteria , nitrogen, phosphorus , Ephemeroptera , wetlands INTRODUCTION shrimp (Palaemonetes paludosus Gibbes), a key food i te m f o r many s pecies of b irds a n d f ish, have b een Nutrient enrichment is a long-standing problem that nearly extirpated from nutrient-enriched areas of this threatens to disrupt the ecological balance of many im- important hemispheric reserve for wildlife (Rader and portant wetlands in the USA and seriously alter the Richardson 1992, 1 994). benefits they provide to society. In the Southeast, for While the end result of excess nutrients can be fairly example, the cumulative effect of excess nutrients has easil y describe d a n d documented, p redicting or d e- resulted in eutrophication of important Atlantic Coast tecting impacts at a stage when management interven- wetland-estuarine systems such as Chesapeake Bay in tion can prevent negative impacts from occurring is Virginia and the Albemarle-Pamlico system in North mor e d ifficult. W etland m anager s n ee d t o p recisely Carolina. Recent outbreaks of a toxic estuarine dino- e valuat e n utrient e nrichmen t wit h t he a id of e arly f lagellat e ( Pfiesteri a p iscicid a S teidinge r a nd B urk- warning tools—bioindicators—for two reasons: (1) to holder), which caused massive kills of recreationally gauge impending effects on wetland biota long before a and economically i mportant f ish and a ffected human catastrophic threshold is reached and (2) to monitor the health, have been attributed to nutrient enrichment of success of efforts to reduce nutrient impacts at lo- cations u p-gradien t s tream s a n d w etland s ( Glasgo w e t a l . where the threshold has been exceeded (i.e., to determine if 1995). The Florida Everglades have undergone exten- best management practices result in mea- surable sive biological changes in response to nutrient inputs improvements). from agricultural activities (Davis 1994). Freshwater Our research evaluates whether growth of filamen- 91
  2. WETLANDS, Volume 20, No. 1, 2000 92 is used to support the road over the floodplain, (2) have similar watershed areas upstream from the cross- i ng s ( 24. 1 a n d 1 8. 4 k m ² f o r B eaverda m a n d K il l Swamps, respectively), (3) are on Bibb-Johnston as- sociation soils (hydric), and (4) have similar propor- tions of area among land uses within their watersheds, with the exception that hog-rearing facilities (a major nutrient source) are present only in the Kill Swamp watershed. Highway crossings, which were construct- ed in 1989, are separated by 1.8 km. Width of per- m anentl y f loode d w etlan d h abita t a t t h e h ighwa y crossings is 200 m and 180 m for Beaverdam and Kill Swamps, respectively. We delineated areas of study as 200 m u pstream a n d downstrea m f rom t he h ighway crossings. Trees of the study wetlands were primarily bald cy- pres s ( Taxodiu m d istichu m (L.) ) a nd s wamp t upelo (Nyssa sylvatica var. biflora Marshall). Macrophyte as- Figur e 1 . L ocatio n o f t h e s tud y w etland s i n t h e c oastal semblages were dominated by the invasive Asian spi- plai n regio n of Nort h Carolina , USA. derwort (Murdannia keisak (Hassk.) Hand.-Mazz.) and rice cutgrass (Leersia oryzoides (L.) Swartz). Duck- wee d ( Spirodel a p olyrrhiz a ( L. ) S chlied. ) wa s a lso tous bacteria on immature aquatic insects can be a use- a bundan t i n o pen-canop y a rea s n ea r t h e h ighwa y ful early-warning bioindicator of detrimental nutrient crossings. These sites were typical of bottomland for- enrichment in wetlands. This technique is an extension e ste d wetland s f ound t hroughou t t he southeast U SA of the method devised by Lemly (1998) for application (Clark and Benforado 1981, Rheinhardt et al. 1998). to streams. Growth of Sphaerotilus sp. and Leptothrix sp. on stream insects has proven to be a useful addition Samplin g Mayfly Abundance. Mayflies were selected to the USEPA Rapid Bioassessment Protocol for Ma- f o r e valuatio n a s a b ioindicato r b ecaus e p reviou s croinvertebrates (Plafkin et al. 1989) because it reveals a stream studies had indicated that this taxon was typi- specific c ause-effec t l inkag e betwee n n utrient e n- cally the most heavily colonized by bacteria and ex- r ichmen t a n d i mpaire d i nsec t c ommunitie s ( Leml y perienced greater impacts (reduction in numbers) than 1998, in press). Practical application of this method is o the r i nsec t g roup s ( e.g. , P lecoptera, T richoptera; quick, simple, and provides for rapid screening of in- L eml y 1998 , i n p ress) . Th e v alidit y o f t hi s i nitial sects in the field. choice was verified for Beaverdam and Kill Swamps The basic premise of the bioindicator (i.e., that bac- by examining three samples of the insect community terial growth reflects nutrient enrichment sufficient to (one collected in October 1995, one in April 1996, and impair insect populations and thus threaten ecosystem one in October 1996). Results of this analysis indicat- i ntegrity ) s houl d b e e quall y t ru e f o r w etland s a n d ed that Ephemeroptera had a greater prevalence and streams. A combination field and laboratory study was i ntensit y o f b acteria l i nfestatio n t ha n o the r i nsec t undertaken to investigate this question and determine groups. Thus, we chose mayflies for use in laboratory if the bioindicator can be applied to wetlands. surviva l e xperiments a s wel l a s f o r a bundanc e e sti- mates in the field. Transect s wer e u se d t o select p lot s f o r s ampling METHODS rather than randomly scattering plots across the entire Field Investigations wetlands because other studies of these swamps had s hown t ha t mayflies were generally more numerous Study Area. The study wetlands, Beaverdam and Kill near the highway crossing (Richardson et al. 1997). Swamps, are third-order watersheds located along In- Thus, to make valid comparisons between swamps, it terstate-40 in Sampson County, North Carolina, USA w a s necessar y t o u s e d at a t ha t were normalize d f or (35° 14' N, 78° 21' W; Figure 1). Both are low-flow- distance. The transect approach addressed that concern ing, cypress-gum wetlands that are part of the Cape but still only provided pseudoreplicates since nutrients Fear River watershed and within the Middle Atlantic and other factors differed between swamps. Transects Coastal Plain Ecoregion (Omernik 1987). These sites were marked parallel to the highway crossings across were selected because both (1) are bisected by fill- the full width of the wetlands. Transects were placed culvert type bridges, where fill dirt rather than pilings
  3. L emly & K ing , I NSECT-BACTERIA B IOINDICATOR 93 Figur e 2. Schemati c view of transect s and samplin g sub-plot s in Beaverda m and Kil l Swamp s in relatio n to the fill-culvert highwa y crossing s on Interstate-40 . Transec t distance s correspon d to distanc e fro m highwa y crossings. at 10, 40, and 200 m distances on both sides of the bucket (600 µm mesh), washed, and preserved with crossing at both wetlands (Figure 2), and three 5 m 95 % e thano l f o r l aborator y p rocessing. A ll mayflies radius sub-plots spaced 40 m apart were placed along were enumerated to genus (no subsampling). each transect. The median sub-plot was placed at the Assessin g B acteria l G rowth . I nsect s were e xamined lowest elevation adjacent to, but not within, the main f o r b acterial growt h u sing a dissectio n microscope ( 10– channel. The remaining two plots flanked the median plot to standardize comparisons among transects. All 200× m agnification). S ome i ndividual s o f e ach plots were inundated with > 10 cm of surface water at mayfly genus were prepared and viewed with scanning the time of sampling. electron microscopy (SEM) using a Philips Model 501 O n M arc h 2 6-27 , 1 997 , m ayfl y a bundanc e w a s instrument. Filamentous bacteria were identified to ge- sampled using protocols developed by the North Car- nus (400–1000× magnification using a compound mi- olina Division of Water Quality (NCDWQ 1997) and c roscope w it h p hase-contras t o ptic s a n d f ibe r o ptic the Mid-Atlantic Coastal Streams Workgroup (USEPA light sources) with identification keys that use external 1997) for low-gradient coastal plain streams. A com- morphological features of the sheaths (e.g., Buchanan posit e sampl e wa s p roduced b y t aking s ubsamples and Gibbons 1974). When present in mature stages, from multiple habitats within a site. We identified four which was the case for bacteria examined in this study, sub-habitats common in all areas of the wetlands: (1) sheath-forming bacteria are easy to identify using sim- herbaceous macrophytes, (2) bald cypress and swamp ple characteristics such as the presence or absence of t upel o t runks , ( 3 ) s ediments , a n d ( 4 ) s ubmerge d i ron or manganese o xide c rust s on s heath s a nd t he woody debris (snags). A D-frame aquatic sweep net presence or absence of swollen tips on sheaths. Even (0.3 m wide, 595 µm mesh) was used to collect sam- preserved material can be used, eliminating the need ples. D-frame sweep nets are the most commonly used for culturing or staining. s amplin g t ool i n strea m a ssessmen t p rotocols ( e.g., The extent of bacterial growth on individual insects F DE P 1 996 , N CDWQ 1 997 , U SEP A 1 997 ) a n d a re was quantified using a block-grid recording technique. useful for estimating community composition in wet- An outline sketch of a generalized representative from lands (Cheal et al. 1993, Turner and Trexler 1997). each order (an enlargement of a line-drawing from a Habitat-specific samples were collected by “ jabbing” a taxonomic key) was copied onto quad-ruled engineer- D-frame sweep net into the target area for a distance of ing paper (25 squares/cm²; each insect ~240 mm long, 0.5 m. One sample was collected from each habitat one insect per page) and used as a data sheet for re- nearest to each sub-plot centroid. Samples were taken cording bacterial growth. An insect was viewed under from all three sub-plots along each transect (12 sam- the microscope, and bacterial growth was recorded by ples per transect). Net contents were placed in a sieve s hadin g t he c orrespondin g body p art on t he s ketch
  4. 94 WETLANDS, Volume 20, No. 1, 2000 with a highlighter pen. A dorsal view and a ventral view were sketched for each individual. The highlight- ed squares in both views were counted and compared to the total number of squares within the outline of the insect to calculate the percent of the body covered by bacteria. Water Quality. Concentrations of dissolved nutrients ( nitrate+nitrite , t ota l N , o rthophosphate , t ota l p hos- phate, in 0.45 µm filtered samples, 4 replicates) were measured when insects were sampled in October 1995, April 1996, October 1996, and April 1997 using meth- ods approved by USEPA for in-situ analysis (USEPA 1992). Grab samples were taken upstream and down- Aq uariu m stream from the highway culvert in each wetland (four upstream, four downstream), filtered in the field, and Figur e 3. Schemati c to p view of aquarium containin g five chambers used to hold mayflies in the survival experiments, immediately refrigerated for transport to the laborato- and a sid e view of a singl e chamber . A tota l of 3 aquaria ry. Nitrate-nitrite concentrations were determined by and 15 chambers was used. copper-cadmium reduction. Total nitrogen concentra- tions were determined by hydrazine reduction follow- ing a persulfate digestion. Nitrogen samples were an- allow w ate r to c irculate f reely bu t smal l e nough t o alyzed on a Traacs 800 spectrophotometer. Orthophos- p reven t i nsect s f rom e scaping . C hamber s wer e s ub- phate and total P concentrations were determined by merged to a depth of 10 cm. t he M urphy-Rile y p hospho-molybdat e blu e c omplex Temperature and pH in aquaria were checked daily reaction. Total P concentrations were determined after and, when necessary, adjusted to maintain conditions p ersulfat e digestion. P hosphorus s ample s wer e mea- similar to the natural swamp (range 14–16°C; pH 5.3– sured using a Beckman DU-64 spectrophotometer. 5.7). A 12 h:12 h 1ight:dark regime was maintained throughout each 30-d experiment. Dissolved nutrient c oncentration s ( nitrate+nitrite , o rthophosphate ) were Laboratory Tests measured on day 1, 10, 20, and 30. Ephemerella sp. and Drunella sp. were selected for During experiments, mayflies fed on algae, diatoms, s tud y b ecaus e ( 1 ) d urin g A pri l a n d O ctobe r ( th e and associated microorganisms that grew as a biofilm month s d urin g whic h l ive mayflie s were c ollected), on the stones in the experimental chambers. Dogwood ( t he y w er e t h e n umericall y d ominan t t ax a i n b ot h Cornus florida L.) leaves were placed among the cob- swamps a nd c ombinin g genera e nsured t ha t e nough ble s t o supplement mayfl y diet s a n d stimulat e t he i ndividual s wer e availabl e f o r t he e xperiments ; ( 2) growth of the biofilm. Leaves were conditioned by in- their herbivorous feeding mode made them amenable cubating them in the chambers for 30 d prior to intro- to long-term laboratory studies; and (3) field collec- ducing the insects. Mayflies were recovered and enu- tions showed that they were heavily colonized by bac- merate d a t t he e nd of e ac h e xperiment a nd percent teria. In April and October 1996 and again in April mortality was determined. Surviving individuals were a n d O ctobe r 1 997 , l iv e E phemerell a a n d D runella examined for bacterial growth under a dissection mi- from each swamp were placed into aerated, polypro- croscope. pylene jars and transported (in a water bath at 15°C to Mayflies were divided into test groups based on the prevent thermal stress on the insects) to Virginia Tech degree of bacterial infestation. Gross visual estimates, University for survival studies. The experiments were rather than the quantitative block-grid procedure used structured to answer two questions: (1) does bacterial for preserved insects, were used to determine the de- growth influence survival and (2) if survival is affect- g re e of bacterial i nfestation. Th e e xperience g ained ed, what levels of infestation are necessary (i.e., what from quantifying bacterial growth on insects from pre- is the threshold) for significant impacts. vious studies (Lemly 1998) made it possible to effi- Three Plexiglas® aquaria with recirculating, aerated, c ientl y s or t mayflie s i nt o g roups. T he f our s urvival and temperature-controlled water supplies were used experiments tested the following levels of infestation: for these experiments (Figure 3). Each aquarium held Experiment (1) two groups—0% and >50% body cov- five 1.5-L chambers (containing several 3-5 cm cob- erage (18 April to 20 May 1996); Experiment (2) three groups bles) into which insects were placed. The sides and —0%, 10–25%, and 25–50% c overage (9 Oc- t obe r b ottoms o f t he c hamber s ha d h ole s l arge e nough t o t o 1 2 N ovembe r 1 996) ; E xperiment ( 3 ) t hree
  5. L eml y & K ing, I NSECT-BACTERIA B IOINDICATOR 95 Tabl e 1. Degre e of bacteria l growt h on th e insec t commu- Tabl e 2. Experimenta l desig n fo r surviva l test s with may- nity of Beaverdam Swamp and Kill Swamp, Sampson Coun- flies. ty, NC. Experimen t 1 (1 8 April–2 0 Ma y 1996) # E xamined Treatments : 0 = n o b acteria l g rowth , X = > 50 % body ( # wit h Bacteria , %) coverage , —   = empt y chamber (# Heavil y Infested* , %) Chamber (5 Mayflies in Each) Samplin g Date Beaverdarn Aquarium # 1 2 3 4 5 and Insec t Order Swam p Kil l s wamp 1 X X 0 0 X Octobe r 1995 2 0 X 0 X 0 Ephemeroptera 3 2 (26,81) (5,16) 2 1 (0) (0) –  3 0 X X 0 Trichoptera 2 0 (3,15) (0) 13 (0) (0) Experimen t 2 (9 October–1 2 Novembe r 1996) Odonata Treatments : 0 = no bacteria l growth , X = 10–25 % body dragonflies 44 (0) (0) 19 (4,21) (1,5) coverage , + = 25–50 % bod y coverage damselflies 25 (21,84) (2,8) 19 (0 ) (0) Chamber (7 Mayflies in Each) Diptera 36 (0 ) (0) 60 (9,15) (0) Hemiptera 27 (18,67) (0) 19 (0) (0) Aquariu m # 1 2 3 4 5 Coleoptera 22 (0) (0) 39 ( 5,13 ) ( 1,3) 1 + 0 X X 0 Apri l 1996 + 2 X + 0 X Ephemeroptera 4 9 ( 41,84 ) (23,47) 63 (0) (0) 3 X + 0 + 0 Trichoptera 1 7 (0) (0) 1 6 (4,25) (0) Experimen t 3 (9 April– 9 Ma y 1997) Odonata Treatments: 0 = 25% of body colonized. X 0 0 X + 3 0 X + + groups—
  6. WETLANDS, Volume 20, No. 1, 2000 96 Figur e 4. Scannin g electro n micrograph s of Ephemerell a sp. illustratin g (a) gill s heavil y infeste d (>25 % covered ) wit h the filamentou s bacteri a Sphaerotilu s sp . an d Leptothri x sp. and (b ) uncolonize d gills. Infestatio n of th e degre e show n in plat e a was associated with 100% mortality in laboratory survival studies and reduced number s of mayfiies in the field. Scale bars = 250 µm. growt h wa s e specially l uxuriant on i nsec t g ills, b ut in Kill Swamp in spring (April) and about 10 times bacterial colonies with similar sheath density occurred greater in fall (October). on all insect body surfaces. Scanning electron micros- Mayfl y A bundance . F ou r g ener a o f m ayflie s w er e c opy i n F igur e 4 d emonstrates t he e xtent t o which c ollected f rom e ac h s wamp ( Tabl e 4) . E phemerella t hese f ilamentou s b acteri a c oul d c olonize individual were most abundant, followed by Drunella, Caenis, gill filaments. a nd Callibaetis . In bot h swamps, mayfl y abundance Under low magnification of the dissection scope, the was greater along transects near the highway crossing bodies of infested insects appeared fuzzy, supporting a (10 m or 40 m) than along the most distant transect light-colored film. Bacterial growth on heavily in- (200 m). The abundance of all four genera was sig- fested (>25% covered) individuals was easily detected nificantly lower in Kill Swamp (Table 4). Caenis sp. with just a hand lens (10–20×) when insects were im- a nd Callibaeti s sp . were a lmost n onexistent i n K ill m erse d i n w ate r o r p reservative . C auda l c erc i o f S wamp bu t were f requently e ncountere d i n s amples Ephemeroptera proved to be particularly good for rap- f rom B eaverdam S wamp . E ve n a mong t he t wo nu- idly screening individuals to assess the degree of bac- merically dominant taxa, there were dramatic differ- terial growth, both in the lab and field (Figure 5). ences in abundance, with 71% fewer Ephemerella sp. A total of six orders of insects were examined for and 75% fewer Drunella sp. in Kill Swamp. Within filamentous bacteria (Table 1). All orders from Beav- t ransects, t here wer e n o significan t u pstream-down- erdam Swamp were free of bacterial growth. In con- stream differences in abundance in either swamp. trast, in Kill Swamp, all orders were colonized by fil- amentous Sphaerotilus sp. and Leptothrix sp. Ephem- eroptera were consistently the most heavily infested, Laboratory Tests with up to 47% of individuals having >25% of their In all 4 experiments, mayflies that supported heavy bodies colonized. Zygoptera also had a relatively high bacterial growth (>25% body coverage) suffered near- p ercentage o f heavil y c olonized i ndividuals . I nfesta- ly 100% mortality within the 30-d experimental run. tion was lowest on Trichoptera, Coleoptera, and Dip- In contrast, mean survivorship among uninfested may- tera. The prevalence and intensity of bacterial infes- flies was 86.5% (±2.1 SE), and these individuals ap- tation was consistent across the three sampling dates. peared healthy (some had grown enough to develop Water Quality. Concentrations of dissolved nutrients wing pads). None of the surviving mayflies that were were consistently greater in Kill Swamp than in Beav- uninfested at the start of the tests became colonized erdam Swamp (Table 3). In particular, the concentra- by bacteria, indicating that there was no chamber-to- tions of nitrate+nitrite and orthophosphate, which are c hambe r g rowt h of b acteria. Concentrations o f d is- n utrient s r esponsibl e f o r m an y b acteria l a n d a lga l s olve d nutrient s ( orthophosphate , nitrate+nitrite ) i n blooms in aquatic systems, were about 5 times greater the aquaria remained below 10 µg/L throughout all of
  7. Figur e 5. Characteristi c appearanc e of bacteria l growt h on cauda l cerc i (tai l filaments ) of Ephemerell a sp . immerse d in 80% ethanol . Plat e a (20 × magnification ) show s uninfeste d cerc i wit h delicate , hair-like seta e visible . Plat e b (20 × magnification) illustrate s the appearanc e of heav y bacteria l growt h (>25 % of bod y covered) . Bacteria l sheath s nearl y fil l the spac e between cerc i and obscur e the delicat e setae . Plat e c (15 × magnification ) show s an advance d stag e of colonizatio n in whic h bacterial filament s hav e becom e matte d and partiall y covere d by sil t particles . Infestatio n of the degre e show n in plate s b and c was associated with 100% mortality in laboratory survival studies and reduced number s of mayflies in the field. The condition can be easil y diagnose d in the field usin g a han d lens with 10–20 × magnification . Scal e bar s = 0. 5 mm. the tests. A plot of the relationship between severity of bacterial infestation and survival of Ephemerella sp. and Drunella sp. revealed that a threshold for cata- strophi c mortalit y e xist s a t a bout t he 25 % l eve l o f body coverage (Figure 6). Almost all individuals with Tabl e 3. Mea n concentration s of dissolve d nutrient s (µg/L) >25% b ody c overag e died, b ut many o f t hose with in Kil l Swam p and Beaverda m Swamp , Sampso n County, 10–25 % c ove rag e s urvi ve d a n d a ppeare d t o b e NC during 1995–97, n = 4. t -probabilities (Kill–Beaverdam healthy. c omparison) : * * * = p < 0 .001 ; – = p aramete r n o t m ea- s ured. Rati o of DISCUSSION Beaver - Kill : Month, Year, and Kil l dam Diagnostic Capability Beaver - t -prob- Nutrient Swamp Swamp dam ability The findings of this study parallel those of Lemly Octobe r 1995 (1998), who concluded that the occurrence of epizoic *** Nitrat e + nitrite 864.7 80.4 10.6 bacterial colonization of aquatic insects can be a use- *** Tota l nitrogen 1926.9 715.0 2.7 ful, quick indicator of detrimental point- or non-point- *** Orthophosphate 119.2 11.1 10.7 source nutrient enrichment in streams. Our study sup- Total phosphat e ports that conclusion in a wetland. The degree of bac- Apri l 1996 terial growth associated with the mortality threshold Nitrate + nitrite can be used as a diagnostic endpoint. When mortality *** Tota l nitrogen 2639.2 780.0 3.4 data from the laboratory experiments are examined in *** Orthophosphate 164.8 34.4 4.8 combination with relationships between mayfly abun- *** Tota l phosphate 243.4 96.8 2.5 dance and bacterial infestation in the field, 20–30% Octobe r 1996 body coverage emerges as a range in which a diag- Nitrat e + nitrite nostic endpoint for the bioindicator can be identified 1.7 *** 1274.7 2162.7 Tota l nitrogen (Figure 6). Survival of insects with 10–25% coverage *** 24.7 11.2 276.0 Orthophosphate c an be g ood, bu t beyon d 30%, s urvival i s u nlikely. 3.1 *** 620.4 198.1 Tota l phosphate Thus, the metric hereafter designated to signify harm- Apri l 1997 ful impacts of nutrients on wetland mayfly populations Nitrat e + nitrite is 25% body coverage by filamentous bacteria. 2.0 *** 1967.7 3888.9 Tota l nitrogen As wit h L emly’s e arlie r f indings , r esult s o f t his *** 17.5 5.7 100.3 Orthophosphate study s eeme d t o i ndicat e a c ause-effec t l inkag e b e- 3.1 *** 54.2 168.8 Tota l phosphate t wee n nutrien t c oncentrations , b acterial growth , a nd
  8. WETLANDS, Volume 20, No. 1, 2000 98 Tabl e 4. Abundance (number collected per transect, refer to Figure 2) of mayflies in Kill Swamp (K) and Beaverdam Swamp (B), Sampson County, NC in March 1997. ** = P < 0.01 (F(1,2) for totals, F(1,5) for  grand   total),   Kill­Beaverdam   comparison. Numbe r of Individual s (ANOV A Result) Callibaetis Ephemerella Drunella Caenis K B Transect K B K B B K Upstream 7 0 9 0 7 29 6 21 10-m 2 0 5 0 4 15 11 37 40-m 0 0 1 0 0 8 4 13 200-m 9 ** 11 52* * 0 15* * 0 Tota l 21 71* * Downstream 7 0 11 1 6 25 9 33 10-m 5 2 6 0 5 18 7 20 40-m 1 0 0 0 3 7 3 12 200-m 13** 2 17** 1 14 50** 19 65** Total 22** 2 32** 1 25 102** 40 136** Gran d Total insect mortality. However, since the laboratory studies growth survived and appeared to be healthy. The abun- used field-infested mayflies, it is not possible to know if dance of mayflies in the study wetlands was signifi- the bacteria alone were responsible for death, or if death cantly lower where nutrients were elevated and bac- was due to a combination of bacteria and other stressors terial growth occurred (Tables 1, 4). Our results show to which the insects were exposed in the field (e.g. , that the insect-bacteria bioindicator can correctly di- t urbidity, f low, dissolved oxygen f luctuations, etc.). a gnose n utrient e nrichment a s a c ause f o r impaired Also, nutrient concentrations in the “clean” wet- land mayfly populations. were sometimes as high (at their peak) as in the enriched wetland (at their low). A bloom stage growth of Reliability and Simplicity Sphaerotilus and Leptothrix can be created when nutrient/temperature relationships reach some critical This is now the third study to confirm experimen- point, but that point is not well defined, even in con- tally that bacterial infestation of insects has practical t rolle d o utdoo r c hannel s ( Phau p a n d G anno n 1 967). application as a bioindicator of detrimental nutrient en- However, up to the critical point, nutrients can be el- richment in a field setting. Two of those studies in- evated and not cause bacterial blooms (Curtis 1969). vestigated streams (Lemly 1998, in press), and one ex- Thus, although there was an overlap of nutrient levels amined wetlands (this paper). As yet, there have been in the two study wetlands, concentrations must have no false positives (i.e., locations in which nutrient en- stayed below the critical point in Beaverdam Swamp richment and bacterial growth occur, but there are no but exceeded that threshold in Kill Swamp. This find- d iscernabl e i mpact s o n m acroinvertebrat e p opula - i ng actuall y strengthen s t he diagnosti c p owe r of t he tions). Thus, the reliability of the method seems suf- bioindicator—it is only evident when detrimental en- ficient to justify further application and investigation, richment occurs. particularly with regard to wetlands. We believe that the evidence for a cause-effect link Importantly , d etection of t he d iagnosti c e ndpoint between nutrients and bacterial growth is strong. Nu- (insects with > 25% body coverage) is easily accom- t rient s wer e s ignificantl y a nd c onsistentl y h ighe r in plished under low magnification (10–20×) with a hand Kill Swamp, which was the only location where bac- l ens o r d issectio n m icroscope ( Figur e 5 ) . D etailed terial growth on insects occurred. Results of the sur- q uantitativ e m easurement s a n d t axonomi c i dentifica- vival studies, in combination with evidence from the tions are not necessary; qualitative samples and order- field surveys, indicate that bacterial growth can have a level classification are adequate. Moreover, insects can major influence on wetland insect populations. For be scanned on-site, literally in-hand, allowing a screen- example, mayflies from the field samples were often ing-level field assessment to be conducted within min- h eavil y c olonize d b y b acteri a ( e.g. , u p t o 4 7 % o f utes. E phemeroptera , T abl e 1 ) . I n t he l aborator y e xperi- Preservatio n of i nsect s i n e thano l or f ormalin, o r ments, < 10% of t he heavil y i nfested mayflie s s ur- manipulation of insects with collection equipment such v ived , w herea s > 85 % o f t hos e w ithou t b acteria l as brushes and forceps apparently does not dislodge
  9. sented here could be useful in the development of a W etland B ioassessment P rotocol ( WBP) o r a m ulti- metric index such as IBI (Index of Biological Integrity; K arr a n d C hu 1 997 ) f o r a pplication t o e cosystems whose macroinvertebrate fauna does not lend itself to evaluation by the classic stream RBP Positive diag- nosis of bacterial growth immediately reveals a prob- a ble c ause f o r i mpaire d wetlan d macroinvertebrate communities, and it can help to focus subsequent in- vestigations because nutrient enrichment is indicated as a major contributing factor. These strengths, com- bine d wit h t he s implicity a n d speed of t he m ethod, suggest that it would be a key element of a WBP or IBI. Degree of bacterial growth (% of body covered) In promoting the use of the bioindicator, we do not imply that Ephemerella or Drunella are the best or Figur e 6 . R elationshi p b etwee n d egre e o f b acteria l i nfes- only taxa to use in a field assessment or that they are tatio n and survival of mayflie s ( Ephemerell a sp . and Dru- assumed to be ubiquitous in wetlands. Our study wet- nella sp.) for 30-d in the laboratory. Each dot represents the lands had a noticeable flow, particularly during periods percent survival from one infestation-level group for one 30- day study (25–35 individuals in the test group, plotted as the of high water, which may account for the dominance mid-rang e of the infestatio n level s for th e test group ; i.e., of these typical stream genera in the samples. Impor- 20–30 % g rou p i s plotte d a s 2 5%) . A t hreshol d f o r c ata- tantly, our results show depression of all mayfly genera strophic mortality exists at about the 25% level of body cov- concurrent with bacterial infestation, including Caenis erage . Beyon d thi s leve l of infestation , ver y few individuals and Callibaetis, which are more typical of the swamp survive. taxa found in the Southeast. We selected Ephemerella and Drunella because they were the only mayflies nu- merous enough to supply the individuals needed for the bacteria. Consequently, severity of infestation can the laboratory experiments. However, we believe that be confirmed in the laboratory without loss of accu- the bioindicator is applicable to a wide variety of wet- racy. Archived samples collected as part of a long-term lands and that mayflies, as a group, are the best taxon m onitorin g p rogra m o r o the r r esearc h p urpose s c an t o u s e i n d etectin g d etrimenta l l evel s o f b acteria l a ls o b e e valuated. Immersing i ndividua l i nsect s into growth. water or preservative suspends bacterial filaments at- tached to the lateral edges of the body for easy rec- ognition, particularly on the caudal filaments of heavi- CONCLUSIONS l y i nfeste d E phemeropter a ( Figur e 5 ) . I ndividual s This study provides evidence that the insect-bacteria whose bodies are   >    25% covered by bacteria (i.e., the bioindicator is valid for application to nutrient assess- indicator level for impact assessment) can be rapidly ment in wetlands. B acterial g rowt h o n i nsect s i s a detected in the field or laboratory. p ractical t ool f or i dentifying t he e xistenc e of d etri- mental n on-point-sourc e n utrient i nputs, a s wel l a s Application to Wetland Bioassessment e valuating t he severit y of biological i mpact s f rom known sources. Rapid field or laboratory screening of In streams, it is possible to use aquatic insects for macroinvertebrate samples is possible. A discovery of rapid assessment of biotic conditions. The EPA Rapid mayflies whose bodies are   >    25% covered by filamen- Bioassessment Protocol for Macroinvertebrates (RBP, tous bacteria is all that is necessary to reliably diag- Plafkin et al. 1989) was developed specifically for that nose harmful impacts of nutrients on wetland macroin- purpose. However, there is no comparable assessment vertebrate communities. The information provided by method for wetlands (Danielson 1998). The e nviron- this bioindicator will be useful for detecting nutrient mental tolerance ratings that form the foundation for problems as well as monitoring the success of man- R BP i n s treams d o no t c onve y t he sam e e cological agement actions to improve water quality. significance when applied to wetlands. For example, a Additional investigations are needed to determine if predominance of species that tolerate warm, turbid wa- the method performs consistently for different types of ter and silty substrate indicates poor biotic conditions wetlands (forested vs. pothole, etc.), nutrients (phos- in upland streams; yet, tolerant species adapted to a phorus vs. nitrogen), and nutrient sources (chemical w id e r ang e o f c ondition s m a y b e c haracteristi c o f fertilizers, industrial and municipal wastewater, animal healthy wetlands. The insect-bacteria bioindicator pre-
  10. 100 WETLANDS, Volume 20, No. 1, 2000 t e s amplin g a n d h abita t a ssessmen t m ethods : 1 . F reshwater wastes), and if the 25% infestation level is the appro- streams and rivers. Prepared by Florida Department of Environ- priate indicator threshold in those cases. There is also a mental Protection, Tallahassee. FL, USA. need to investigate why some insect taxa are consis- tently Glasgow, H. B. Jr., J. M. Burkholder, D. E. Schmechel, P.  A. Tester, and P.   A. Rublee. 1995. Insidious effects of a toxic estuarine di- more heavily colonized by bacteria than others, i.e., noflagellate on fish survival and human health. Journal of Toxi- factors such as differences in physiological param- eters cology and Environmental Health 46:501–522. (chemical composition of body surfaces) or eco- l ogical Karr, J. R. and E. W. Chu. 1997. Biological monitoring and assess- v ariable s ( degre e o f movement , m icrohabitat usage, ment: using multimetric indexes effectively. USEPA, Office of Research and Development. Washington, DC, USA. EPA 235- etc.). Finally, because mayflies are not common in all R97-001. wetlands, other taxa (e.g., odonates) should be investigated Lemly, A. D. 1998. Bacterial growth on stream insects: potential for as a possible surrogate for diagnosing nu- trient use in bioassessment. Journal of the North American Bentholog- ical Society 17:228–238. impacts. Lemly, A. D. In press. Using bacterial growth on insects to assess nutrient impacts in streams. Environmental Monitoring and As- sessment. ACKNOWLEDGMENT S North Carolina Division of Water Quality (NCDWQ). 1997. Stan- dard operating procedures, biological monitoring. North Carolina We thank Kevin Nunnery for assistance with field Department of Environment and Natural Resources. Raleigh, NC, s amplin g an d w ate r q ualit y analyse s a nd H olly J en- U SA. Omernik, J. M. 1987. Ecoregions of the conterminous United States. nings for assistance with the laboratory experiments. Annals of the Association of American Geographers 77:118–125. D r . J on L ewi s a nd t he D epartment o f P athology a t Phaup, J. D. a nd J. Gannon. 1967. Ecology of Sphaerotilus in an Bowman Gray School of Medicine, Wake Forest Uni- experimental outdoor channel. Water Research 1:523–541. Plafkin, J. L., M. T Barbour, K. D. Porter, S. K. Gross, and R. M. versity, Winston-Salem, North Carolina provided fa- Hughes. 1989. Rapid bioassessment protocols for use in streams cilities and assistance with scanning electron micros- and rivers: benthic macroinvertebrates and fish. U.S. Environmen- copy. The images for Figures 4–5 were prepared by t a l P rotectio n A gency , O ffic e o f R esearc h a nd Development, Washington, DC, USA. EPA 444/4-89-001. PhotoGraphic Services, Virginia Tech University. Drs. Rader, R. B. a nd C. J. Richardson. 1992. The e ffects of nutrient Curti s R ichardso n an d A ndre w D olloff p rovide d r e- enrichment on algae and macroinvertebrates in the Everglades: a view comments that improved the paper. review. Wetlands 12:121–135. Rader, R. B. and C. J. Richardson. 1994. Response of macroinver- tebrates and small fish to nutrient enrichment in the northern Ev- erglades. Wetlands 14:134–146. LITERATURE C ITED Rheinhardt, R. D., M. C. Rheinhardt, M. M. Brinson, and K. Faser. 1998. Forested wetlands of low order streams in the inner coastal Buchanan, R. E. and N. E. Gibbons. 1974. Bergey’s Manual of De- plain of North Carolina, USA. Wetlands 18:365–378. terminative Bacteriology. Williams and Wilkins. Baltimore, MD, Richardson, C. J., R. S. King, and K. Nunnery. 1997. A functional U SA. assessment of wetland response to highways: Phase I macroin- Cheal, F., J. A. Davis, J. E. Crowns, J. S. Bradley. and F.  H. Whittles. vertebrate community studies. Duke University Wetland Center, 1993. The influence of sampling method on the classification of Durham, NC. USA. Publication 97-02. wetland macroinvertebrate communities. Hydrobiologia 257:47– nd Sokal, R. R. and E J. Rohlf. 1981. Biometry. 2 edition. W.H. Free- 54. man and Co., San Francisco, CA, USA. Clark, J. R. and J. Benforado (eds.). 1981. Wetlands of Bottomland Turner, A. M. and J. C. Trexler. 1997. Sampling aquatic invertebrates Hardwood Forests. Elsevier, Amsterdam, The Netherlands. from marshes: evaluating the options. Journal of the North Amer- Curtis, E. J. C. 1969. Sewage fungus: its nature and effects. Water ican Benthological Society 16:694–709. Research 3 :289–311. United States Environmental Protection Agency (USEPA). 1992. Danielson, T. J. 1998. Wetland Bioassessment Fact Sheets. U.S. En- Methods for chemical analysis of water and wastes. USEPA, Of- vironmental Protection Agency, Office of Wetlands, Oceans, and fice of Research and Development, Washington. DC, USA. Watersheds. Wetlands Division, Washington, DC, USA. EPA 843- United Stated Environmental Protection Agency (USEPA). 1997. F-98-001. Field and laboratory methods for macroinvertebrate and habitat Davis, S. M. 1994. Phosphorus inputs and vegetation sensitivity in assessment of low gradient, nontidal streams. Mid-Atlantic Coast- the Everglades. p. 357–378. In S. M. Davis and J. C. Ogden (eds.) al Streams Workgroup, Environmental Services Division, Region Everglades: The Ecosystem and Its Restoration. St. Lucie Press, 3, Wheeling, WV, USA. Delray Beach, FL, USA. Florid a D epartmen t o f E nvironmenta l P rotectio n ( FDEP) . 1 996. Manuscript received 4 February 1999; revisions received 29 April Standard operating procedures manual—benthic macroinvertebra- 1999 and 9 August 1999; accepted 4 November 1999.



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