Kadmiumaltistuksen havaitseminen Hydropsyche pellucidula -vesiperhostoukilla (Trichoptera: Hydropsychidae) hyperspektrikuvantamisen ja kidusvaurioiden avulla
Metallipäästöt aiheuttavat vesistöissä eliöyhteisöille monenlaisia haittavaikutuksia, joita voidaan tutkia bioindikaattori- eli ilmentäjälajimenetelmin. Vesiperhostoukkia (Trichoptera) on käytetty virtavesien metallisaastumisen indikaattoreina, sillä ne kestävät metallialtistusta suhteellisen hyvin, mutta altistuksen seurauksena toukilla voi esiintyä morfologisia muutoksia, kuten kidusvaurioita tai pintakudoksen tummumista. Koska toisaalta vierasaineiden kudospitoisuusmittaus on usein työlästä ja toisaalta rakenteellisten muutosten arviointi altis subjektiiviselle harhalle, tavoitteenani oli kehittää objektiivinen ja kustannustehokas tapa selvittää metallialtistusta ja sen vaikutuksia pohjaeläimistä. Hyperspektrikuvantamisella (hyperspectral imaging, HSI) voidaan tutkia aineiden pitoisuusvaihteluja eri matriiseissa. Sitä hyödynnetään jo muun muassa maaperätutkimuksessa, maataloudessa ja elintarviketeollisuudessa. Tutkielmassani tutkin hyperspektrikuvantamisen soveltuvuutta kadmiumin havaitsemiseen Hydropsyche pellucidula -vesiperhostoukista. Toukkia altistettiin kadmiumkloridille 96 tuntia subletaaleissa pitoisuuksissa 9,9, 94,5, 880 ja 8996 µg Cd l-1. Toukat kuvattiin HSI-kameralla 500–665 nm aallonpituuksilla, niistä tutkittiin mikroskoopilla kidusten vauriot ja niiden Cd-pitoisuudet määritettiin. Kudosten ja altistusveden kadmiumpitoisuuden välillä oli positiivinen korrelaatio (Spearman, n = 74, r = 0,93, p < 0,001). Kidusvaurioita esiintyi kaikissa altistuspitoisuuksissa, mutta eniten pitoisuudessa 94,5 µg l-1. Kudosten kadmiumpitoisuudella ja kidusvaurioiden esiintymisellä ei havaittu yhteyttä (Spearman, n = 74, r = -0,034, p = 0,774). Hyperspektriaineiston perusteella toukkien hemolymfan trehaloosi- eli disakkaridipitoisuus korreloi negatiivisesti (Pearson, n = 30, r = -0,43, p < 0,05) kudosten kadmiumpitoisuuden kanssa. Takaruumiin tumma pigmentti, eumelaniini, runsastui (Pearson, n = 30, r = 0,61, p < 0,001) ja vaalea pigmentti, feomelaniini, väheni (Pearson, n = 30, r = -0,62, p < 0,001) kuvan spektrin kokonaisintensiteetin kasvaessa. Kudosten kadmiumpitoisuus nähtävästi vaikutti kokonaisintensiteettiin ja pigmenttisuhteisiin välillisesti, sillä kudospitoisuudella ei havaittu yhteisvaihtelua näiden muuttujien kanssa. Havaitut spektripiirteiden muutokset toukissa viittaavat stressi- tai immuunireaktioihin altistuksen seurauksena. Hyperspektrikuvantaminen vaikuttaa lupaavalta menetelmältä kadmiumaltistuksen havaitsemiseen, mutta sen laajempi käyttöönotto vaatii sisäänajoa eri lajeilla ja metalleilla.
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Detecting cadmium exposure from Hydropsyche pellucidula
(Trichoptera: Hydropsychidae) larvae using hyperspectral
imaging and incidence of gill anomalies.
The effects of metal pollution on aquatic biota can be studied using bioindicator methods. Caddis larvae (Trichoptera) are widely used to indicate metal contamination of stream waters since they are relatively tolerant to metal exposure, but show morphological responses, such as gill anomalies or darkening of surface tissue. Because the analyses of tissue concentrations are often quite laborious and evaluation of morphological responses liable to subjective error, my goal was to develop an objective, cost-efficient method to detect metal exposure and its effects from benthic macroinvertebrates. Hyperspectral imaging (HSI) can be used to detect concentrations of elements and substances in different matrices. HSI has applications already in soil research, agriculture and food industry. In this study, I tested the applicability of HSI in detecting sublethal Cd exposure of Hydropsyche pellucidula larvae. Larvae were exposed to CdCl2 for 96 hours, in Cd concentrations of 9.9, 94.5, 880 ja 8996 µg Cd l-1. The measured endpoints were the Cd-concentration of larvae and occurrence of gill anomalies. All larvae were imaged using 500-665 nm visible light wavebands. The measured tissue Cd concentration of the larvae correlated significantly with the exposure concentration (Spearman, n = 74, rs = 0.93, p < 0.001). Gill anomalies were present at all exposure concentrations, but were the most frequent at 94.5 µg Cd l-1. However, there was no association between the incidence of gill anomalies and the Cd tissue concentration (Spearman, n = 74, r = -0.034, p = 0.774). Based on the HSI results, the trehalose concentration (a disaccharide) in larval hemolymph decreased with increasing Cd tissue concentration (Pearson, n = 30, r = -0.43, p < 0.05). Pigmentation of larval abdomen changed as the total intensity of images increased; the relative amounts of eumelanin, a dark pigment, increased (Pearson, n = 30, r = 0.61, p < 0.001) and pheomelanin, yellow pigment, decreased (Pearson, n = 30, r = -0.62, p < 0.001) with increasing total intensity. It seems that tissue Cd concentration affected total intensity and pigment proportions indirectly, since tissue concentration did not correlate with these variables. The observed changes in spectral features suggest stress or immune reactions followed by Cd exposure. Based on the results, hyperspectral imaging is a promising technique for detecting Cd exposure from H. pellucidula, though more research with different species and metals is needed.
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