Acta Biologica 26/2019

Acta Biologica 26/2019 (dawne Zeszyty Naukowe Uniwersytetu Szczecińskiego Acta Biologica) Rada Redakcyjna / Editorial Board Vladimir Pésič (University of Montenegro) Andreas Martens (Karlsruhe University of Education) Stojmir Stojanovski (Hydrobiological Institute in Ohrid) Clémentine Fritsch (Université de Franche-Comté) Leszek Jerzak (University of Zielona Góra) Pedro Jiménez-Mejías (Department of Botany, Smithsonian Institution, National Museum of Natural History) Francesco Spada (University of Rome) Barbara Płytycz (Jagiellonian University in Krakow) Joana Abrantes (Universidade do Porto) Pedro J. Esteves (Universidade do Porto)

Recenzenci / Reviewers Prof. Vladimir Pésič – University of Montenegro (Montenegro), prof. Alireza Saboori – University of Tehran (Iran), prof. Lateef O. Tiamiyu – University of Ilorin. (Nigeria), prof. Adetola Jenyo-Oni – University of Ibadan (Nigeria), prof. Mohsen Abdel-Tawwab – Agricultural Research Center (Egypt), prof. Andrzej K. Siwicki – Uniwersytet Warmińsko Mazurski (Polska), prof. Michał Stosik – Uniwersytet Zielonogórski (Polska), prof. Seban Proches – University of KwaZulu-Natal (RPA)

Redaktor naukowy / Editor dr hab. Dariusz Wysocki

Sekretarz redakcji / Secretary Editor dr hab. Helena Więcław

Korektor / Proofreader Elżbieta Blicharska

Redakcja techniczna i skład komputerowy / Technical editorial and Text Design Wiesława Mazurkiewicz

Projekt okładki / Cover design Bartosz Łukaszewski

Czasopismo jest indeksowane / The journal is indexed Baza AGRO, Index Copernicus

Pełna wersja publikacji / Full version of publication available www.wnus.edu.pl/ab

Wersja papierowa jest wersją pierwotną / Paper version of the journal is an original version © Copyright by Uniwersytet Szczeciński, Szczecin 2019

ISSN 2450-8330 (dawne 1230-3976; 1640-6818)

WYDAWNICTWO NAUKOWE UNIWERSYTETU SZCZECIŃSKIEGO Wydanie I. Ark. wyd. 10,0. Ark. druk. 8,3. Format B5. SPIS TREŚCI / CONTENTS

Ibrahim Adeshina, Benjamin O. Emikpe, Adetola Jenyo-Oni, Emmanuel K. Ajani, Musa I. Abubakar Growth performance, gut morphometry and innate immune profiles of common carp, Cyprinus carpio juveniles fed diet fortified with Mitracarpus scaber leaves extract and its susceptibility to pathogenic bacteria, Aeromonas hydrophila 5–17

Musa I. Abubakar, Ibrahim Adeshina, Ikililu Abdulraheem, Somira Abdulsalami Histopathology of the gills, livers and kidney of Clarias gariepinus (Burchell, 1822) exposed to sniper 1000EC under laboratory conditions 19–30

Furkan Durucan First record of Thecacineta calix (Ciliophora: Suctoria) on harpacticoid copepod from Aegean Sea, Turkey 31–34

Małgorzata Pawlikowska-Warych, Beata Tokarz-Deptuła, Paulina Czupryńska, Wiesław Deptuła Biofilm and Quorum Sensing in Archaea 35–44

Małgorzata Pawlikowska-Warych, Paulina Czupryńska, Beata Tokarz-Deptuła, Wiesław Deptuła Somatic and F-specific bacteriophages in waters of the small, municipal Rusałka Lake in Szczecin 45–55

Marina Patsyuk Changed species composition of naked amoebae in soils of forest-and-steppe zone of Ukraine 57–64

Mona Moghadasi, Azadeh Zahedi Golpayegani, Alireza Saboori, Hossein Allahyari, Hamideh Dehghani Tafti Tetranychus urticae changes its oviposition pattern in the presence of the predatory mites, Phytoseiulus persimilis and Typhlodromus bagdasarjani 65–81

Dominika Bębnowska, Paulina Niedźwiedzka-Rystwej Characteristics of a new variant of rabbit haemorrhagic disease virus – RHDV2 83–97

Robert Stryjecki A faunistic and ecological characterization of the water mites (Acari: Hydrachnidia) of the Branew River (central-eastern Poland) 99–115 Tapas Chatterjee Scanning electron microscopic observation of Acarothrix grandocularis (Acari, Halacaridae) and notes on the species of the genus Acarothrix 117–126

Tapas Chatterjee, Mandar Nanajkar Report of Rhombognathus scutulatus (Acari: Halacaridae) from Goa, India 127–132

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Acta Biologica 26/2019 | www.wnus.edu.pl/ab | DOI: 10.18276/ab.2019.26-01 | strony 5–17

Growth performance, gut morphometry and innate immune profiles of common carp, Cyprinus carpio juveniles fed diet fortified with Mitracarpus scaber leaves extract and its susceptibility to pathogenic bacteria, Aeromonas hydrophila

Ibrahim Adeshina,1 Benjamin O. Emikpe,2 Adetola Jenyo-Oni,3 Emmanuel K. Ajani,4 Musa I. Abubakar5

1 Department of Aquaculture and Fisheries, Faculty of Agriculture, University of Ilorin, P.M.B. 1515 Ilorin, Kwara State, Nigeria, ORCID: 0000-0002-2404-2956 2 Department of Veterinary Pathology, Faculty of Veterinary Medicine, University of Ibadan, P.M.B. 01, Ibadan, Oyo State, Nigeria, ORCID: 0000-0003-2458-6504 3 Department of Aquaculture and Fisheries Management, Faculty of Renewable Natural Resources, University of Ibadan, P.M.B. 01, Ibadan, Oyo State, Nigeria, ORCID: 0000-0002-2589-1387 4 Department of Aquaculture and Fisheries Management, Faculty of Renewable Natural Resources, University of Ibadan, P.M.B. 01, Ibadan, Oyo State, Nigeria 5 Department of Aquaculture and Fisheries Management, Faculty of Agriculture, University of Ilorin, P.M.B. 1515, Ilorin, Kwara State, Nigeria

Corresponding authors, e-mail: [email protected], [email protected], [email protected], [email protected], [email protected]

Keywords Common carp, feed supplements, growth performance, gut morphometry, immunostimulants Abstract Mitracarpus scaber, an endemic medicinal plant to Nigeria, Africa, has medicinal value. In the present study, Mitracarpus scaber leaves extract (MSLE) was fed to common carp, Cyprinus carpio to evaluate its effect on growth performance, nutrient utilization, gut morphometry, and innate immunity parameters. Four isonitrogenous diets (32% crude protein) containing 0.0, 5, 10, or 15 g MSLE/kg diet were fed to fish (7.52 ±0.23 g) for 12 weeks. After the feeding trial, fish were exposed to pathogenic bacteria Aeromonas( hydrophila) for 14 days. Growth performance, nutrient utilization, and feed intake were significantly improved with increasing MSLE levels up to 10 g/kg diet. Similarly, fish fed MSLE diets increased significantly intestinal villi length/width, and absorption area. Furthermore, activities of respiratory burst, lysozyme, catalase, and superoxide dismutase were significantly higher in fish fed diets containing MSLE levels, and their highest values were obtained at fish fed 15 g MSLE/kg diet. After bacterial challenge, fish mortality was lowest (8.45 ±1.30%) in fish fed 15 g MSLE/kg diet, whereas highest mortality (52.50 ±4.56%) was observed with fish fed the control diet. The present study conjured that MSLE inclusion in fish diets with optimum level of 10 g/kg diet stimulated significantly the performance, nutrient utilization, modified gut morphometry, and innate immune response of common carp. Also, its inclusion protected fish against pathogenic bacteria, A. hydrophila infection.

5 Ibrahim Adeshina, Benjamin O. Emikpe, Adetola Jenyo-Oni, Emmanuel K. Ajani, Musa I. Abubakar

Szybkość wzrostu, morfometria jelit i wrodzony profil odpornościowy młodocianych osobników karpia Cyprinus carpio karmionych dietą wzbogaconą ekstraktem z liści Mitracarpus scaber oraz ich podatność na bakterię chrobotwórczą Aeromonas hydrophila Słowa kluczowe karp, wzbogacanie diety, szybkość wzrostu, morfometria jelit, immunostymulanty Streszczenie Mitracarpus scaber, endemiczna roślina lecznicza z Nigerii w Afryce, ma wartość leczniczą. W niniejszym badaniu ekstrakt z liści Mitracarpus scaber (MSLE) podawano karpiowi, Cyprinus carpio, aby ocenić jego wpływ na szybkość wzrostu, wykorzystanie składników odżywczych, morfometrię jelit i parametry odporności wrodzonej. Cztery izoazotowe diety (32% surowego białka) zawierające 0,0; 5, 10 lub 15 g diety MSLE/kg podawano młodym rybom (7,52 ±0,23 g) przez 12 tygodni. Po tym okresie ryby były narażone na bakterie chorobo- twórcze (Aeromonas hydrophila) przez 14 dni. Szybkość wzrostu, wykorzystanie składników odżywczych i spożycie paszy zwiększały się wraz ze wzrostem poziomu MSLE do wartości 10g/kg diety. Ryby ze wzbogaconą dietą znacznie zwiększyły stosunek długości/szerokości kosmków jelitowych i obszar wchłaniania. Ponadto wielkość wybuchu tlenowego, oraz aktyw- ność lizozymu, katalazy i dysmutazy ponadtlenkowej były znacznie wyższe u ryb karmionych pokarmem wzbogaconym o MSLE. Najwyższe wartości wyżej wspomnianych parametrów uzyskano u ryb karmionych pokarmem wzbogaconym o 15 g MSLE/kg. Po wprowadzeniu bakterii śmiertelność ryb była najniższa (8,45 ±1,30%) u ryb karmionych 15 g diety MSLE/ kg, podczas gdy najwyższą śmiertelność (52,50 ±4,56%) obserwowano u ryb karmionych dietą kontrolną. W niniejszym badaniu wykazano, że włączenie MSLE w ilości 10 g/kg diety zna- cząco stymuluje wydajność, wykorzystanie składników odżywczych, modyfikuje morfometrię jelit i wrodzoną odpowiedź immunologiczną karpia. Włączenie do diety MSLE chroniło ryby przed infekcją bakteriami chorobotwórczymi, A. hydrophila.

Introduction

Fish are considered as ones of the highly nutritional sources for animal proteins. It has high- quality protein, vitamins and minerals, and unsaturated fatty acids. The knowledge of nutritional, and health benefitof fish has resulted into its consumption (Adeshina, Jenyo-Oni, Emikpe, Ajani, Abdel-Tawwab, 2018b); therefore, creating mammoth gap between the demand, and supply for fish.In recent times, aquaculture has been augmenting shortage between the demand, and supply of fish, following the shortageof supply from captured fisheries. However, diseases are impeding the success of aquaculture industry. Series of researches have been focused on use of phytobiotics to combat fish diseases, and improve their immunity (Abdel-Tawwab, 2010, 2012, 2015, 2016; Abdel-Tawwab, Abbass, 2017; Abdel-Tawwab, Ahmad, 2009; Guardiola et al., 2016; Van Doan, Hoseinifar, Dawood, Chitmanat, Tayyamath, 2017; Hoseinifar, Dadar, Khalili, Cerezuela, Esteban, 2017a; Hoseinifar et. al. 2017b; Adeshina, Adewale, Tiamiyu, 2017; Abdel-Tawwab, Adeshina, Jenyo-Oni, Ajani, Emikpe, 2018a). This is because medicinal plants such as Mitracarpus scaber are generally regarded as safe (GRAS), have no residual effect, environmental eco-friendly among other factors.M. scaber belongs to the family Rubiaceae with about 30 different species. However, in Nigeria, M. scaber is the commonest species. The leaves of this plant are used to treat many diseases in both man and animal. It has been reported to have antimicrobial and antimycotic properties such as gallic acid, 3,4,5-trimethoxybenzoic acid, 4-Methoxyacetophenone, 3,4,5-trimethoxyacetophenone, n-octane, 2-hexanol, p-cymene, α and β pinene etc (Owolabi, Arhewoh, Innih, Anaka, Monyei, 2014). It is claimed that this plant has also antibacterial and antifungal activities (Bisignano et al., 2000; Abere, Onwukaeme, Eboka, 2007; Anejionu et al., 2011).

6 Growth performance of Common carp fed MSLE-fortified diet

Fish production is hindered by disease outbreaks of aeromonoid caused by Aeromonas hydrophila infection. The disease is causing severe loss in fish farming. Some of the signs of aeromonoid include furunculosis, hemorrhage, and red sore disease, among others, leading to mass mortality of fish, and economic losses (Shoko, Limbu, Mgaya, 2016; Talpur, 2014; Tan et al., 2018). The common way of controlling A. hydrophila infection is using antibiotics, which has been discouraged due to evolving disease resistance, environmental degradation among others. Therefore, there is need to focus on developing feed supplements as substitutes to chemothera- peutics for control, and management of bacterial diseases in order to sustain environmentally eco-friendly aquaculture. Common carp is one of the cultured fish species in tropical Africa, and widely accepted (Adeshina, Jenyo-Oni, Emikpe, Ajani, 2018a). To large extent, it is important to develop feed supplements as a replacement to the use of synthetics drugs to promote, and sustain responsible aquaculture. Therefore, the present study was carried out to evaluate the application of Mitrascarpus scaber leaf extract (MSLE) in practical diets for common carp as growth, nutrient utilization, and immune parameters. Fish resistance to pathogenic bacteria, A. hydrophila, was also investigated.

Materials and Methods

Plants collection and identification The fresh leaves of M. scaber were collected from Ile-Apa community, and authenticated in the Herbarium Unit, Forest Research Institute of Nigeria, Ibadan, Nigeria.

Preparation of experimental diets The leaves were dried at 35°C, milled into fine powder and 5 grams were extracted in ethyl- acetate (50 mL) using hot method for 4 hr. The chemical was eliminated in a rotary evaporator at 45°C. The extracts were then stripped into sterilized bottles, and stored at 20°C until use. Four isonitrogenous diets (32% crude protein) were formulated with fish meal (72%), toasted soybean (46.2%), and white maize (9.3%) (CFA, 1974; Drury et al., 1967) to contain 0.0, 5, 10, or 15 g MSLE/kg diet (Table 1).

Table 1. Ingredients and proximate chemical composition (%; on dry matter basis) of the experimental diets containing graded levels of Mitrascarpus scaber leaves extract (MSLE)

MSLE levels (g/kg diet) Ingredients 0.0 (control) 5.0 10.0 15.0 1 2 3 4 5 Fish meal 252.4 252.4 252.4 252.4 Soybean 232.4 232.4 232.4 232.4 Maize 465.2 460.2 455.2 450.2 Starch 15.0 15.0 15.0 15.0 Vegetable oil 15.0 15.0 15.0 15.0 Premixa 20.0 20.0 20.0 20.0 MSLE 0.0 5.0 10.0 15.0 Total 1000.0 1000.0 1000.0 1000.0

7 Ibrahim Adeshina, Benjamin O. Emikpe, Adetola Jenyo-Oni, Emmanuel K. Ajani, Musa I. Abubakar

1 2 3 4 5 Proximate composition (%) Crude protein 32.01 32.04 32.22 32.23 Moisture 9.17 8.53 8.83 9.17 Ether extract 9.39 9.55 9.48 9.90 Ash 8.35 8.78 8.55 8.90 CHO(%)b 28.18 28.10 28.45 27.27 Fibre (%) 12.90 13.00 12.47 12.53 a Premix = HI-MIX®AQUA (Fish) each one kilogram (1 kg) contains; vitamin A, 4,000,000 International Unit (IU); vitamin D3, 8,00,000 IU; vitamin E, 40, 000 IU; vitamin K3, 1,600 mg; vitamin B1, 4,000 mg; vitamin B2, 3,000 mg; vitamin B6, 3,800 mg; vitamin B12, 3 mcg; Nicotinic acid 18,000 mg; Pantothenic acid, 8,000 mg; Folic acid, 800 mg; Biotin, 100 mcg; Choline chloride 120,000 mg; Iron, 8,000 mg; Copper, 800 mg; Manganese, 6,000 mg; Zinc, 20,000 mg; Iodine, 400 mg; Selenium, 40 mg; Vitamin C C(coated), 60,000 mg; Inositol, 10,000 mg; Colbat, 150 mg; Lysine, 10,000 mg; Methionine, 10,000 mg; Antioxidant, 25,000 mg. b CHO = Carbohydrate.

Experimental procedure and design Common carp, C. carpio juveniles were obtained from a reputable farm, and acclimated to lab conditions for 14 days during which fish fed a commercial diet (32% crude protein). Fish (7.52 ±0.23 g) were randomly allotted into 12 aquaria (50 L) at a density of 20 fish/aquarium. The fish were fed on one of the formulated diets up to nearly satiation two times every day at 08:00 am and 06:00 pm for 84 days. Throughout the experiment, water from tanks was replaced every 3-day intervals. Water samples were collected from the rearing tanks to monitor water quality. Water temperature ranged from 23.23 to 25.17°C, dissolved oxygen from 5.80 to 6.66 mg/l, conductivity from 0.36 to 0.90 ms.cm-1, nitrite from 0.95 to 1.00 ppm, nitrate ranged from 33.35 to 53.20 ppm, and pH from 7.15 to 7.89 measured with mercury thermometer, Winkler method, commercial kits (Colombo nitrate, nitrite, and pH test kit) respectively.

Growth performance and nutrient utilization

Fish per each tank were collected, counted, and group-weighed biweekly and at the end of the feeding trial on a digital ScoutPro sensitive scale (Model: KD-200-110, USA). Parameters of growth performance and nutrients utilization were calculated as:

Body weight gain ( g) = W10 − W ; LnW− LnW Specfic growth rate ( SGR; %g / day) = 10 ×100; Length of the culture period Feed intake= summation of feed fed to fish in each tanks / fish No.; Dry weight of feed fed ( g) Feed conversion ratio ( FCR) = ; Fish weight gain ( g)

Wet body weight gain ( g) Protein efficiency ratio ( PER) = ; Crude protein fed

8 Growth performance of Common carp fed MSLE-fortified diet

Protein ( %) in diet × diet consumed Where protein fed = ; 100 Final number of fish stocked Survival rate ( %) = ×100. Initial number of fish stocked

Where: W1 = final mean weight; 0W = initial mean weight; L1 = final mean length; 0L = initial mean length.

Gut morphometry Three fish from each tank were collected and anthesized with buffered tricaine methane sulfonate (30 mg/L). Fish intestine was taken for histological examination. The guts were prepared on slides as described by Drury, Wallington and Roy (1967). The guts were excised, and cut with a sharp scalpel blade into small pieces, and fixed by immersing in modified Bouin’s fluid made up of picric acid (300 mL), formalin (100 mL), and 1% tricarboxylic acid (20 mL) according to the methods of Eyarefe, Emikpe and Arowolo (2008), followed by the dehydration of the tissues in 70, 80, 90, 95%, and absolute ethanol. The sections were prepared (4 mm thickness), followed by hydration in absolute ethanol, 95, 90, 80, and 70% ethanol. Villus length and width (mm), depth of the crypt (DC, mm), villus width (VW, mm), and area of absorption (AA, mm2) were Measurements of taken using light microscope (HE x40) (Olympus CX21, Japan) in triplicates with a micrometer rule (Eyarefe et al., 2008; Fox et al., 1997; Bello et al., 2012).

Area of absorption ( mm2 ) = Villus length( mm)× Villus width( mm)

Innate immunity assays

Three fish from each tank was collected, and anthesized with buffered tricaine methane sulfonate (30 mg/L). Blood collected from the caudal vein and used to determined innate immune parameters within the aid of Randox® Laboratories diagnostic kits (Crumlin, County Antrim, UK). Blood of fish from each experimental unit was pooled together. Superoxide dismutase (SOD) activity was determined by ferricytochrome-C method. Xanthine oxidase was used as the source of superoxide radicals. The quantity of enzyme required to produce 50% inhibition of ferricytochrome-C was taken as one activity unit (McCord, Trouslade, Ryu, 1984). The rate of H2O2 at 240 nm was used to estimate the catalase (CAT) activity (Aebi, 1984). To determine the lysozyme activities in the fish sera, Micrococcus luteus (0.60 mg/mL) was spread in agarose gel (1%), and phosphate buffer (pH 6.2, 50 mM) (Difco BD Co, Franklin Lakes, NJ). Nutrient Agar prepared plates were dug to have wells of 6 mm diameter using cork-borer. The wells were filled with fish sera (25 μL/well), and incubated (25°C) for 20 hr. Then, the lysozyme activity was estimated from a standard curve prepared with lysozyme from chicken egg white (Abdel-Tawwab et al., 2018a). Respiratory burst activity (RBA) was measured with the aid of Randox® Laboratories diagnostic kits (Crumlin, County Antrim, UK) as described by Chiu, Guu, Liu, Pan, Cheng (2007). Nitroblue tetrazolium (NBT) assay was used to measure the RBA which indicates the quantity

9 Ibrahim Adeshina, Benjamin O. Emikpe, Adetola Jenyo-Oni, Emmanuel K. Ajani, Musa I. Abubakar of intracellular oxidative free radicals (Grinde, 1989; Secombes, 1990). Microplate reader (Optica, Mikura Ltd, UK) was used evaluate the NBT reduction.

Bacterial challenge test The susceptibility of common carp, C. carpio was examined using pathogenic bacteria (A. hydrophila) after the 12-week feeding trial. The A. hydrophila (ATCC 4356) isolate was pob- tained from Department of Microbiology Laboratory, University of Ibadan, Nigeria. The LD50 (lethal dose) of the pathogenic bacteria was determined. Bacterial isolates were grown on nutrient broth plus yeast extract, and incubated at 35°C for 24 h to revive the bacteria. Ten (10) fish from each experimental unit were exposed to A. hydrophila, which were grown on nutrient broth plus yeast extract for 24 hour at 35°C in an incubator. Bacterial cells were then centrifuged at 3000 g for 30 min to form pellets. The pellets were resuspended in 1.0 mL of 0.1% peptone water. For the challenge test, fish from each treatment were collected, and 20 fish (10 fish per tank) were transferred into two other tanks previously filled with dechlorinated freshwater as two replicates. Fish were challenged with a 0.1-mL dose of A. hydrophila (5 × 105 CFU/mL) by intraperitoneal injection, and were returned to the experimental setup (Adeshina et al., 2018b; Abdel-Tawwab et al., 2018a). Fish were fasted for 24 h before infection, and refeeding with the corresponding experimental diets 12 h later. All fish groups were kept under observation for 14 days to record any abnormal clinical signs and daily mortality.

Statistical Analysis

The data obtained were subjected to one-way analysis of variance (ANOVA) using IBM statistical package (SPSS version 20) to determine differences among the treatments in all parameters. Individual means were separated using Duncan multiple range test. Quadratic regression was used to determine the optimum level of extract for weight gain. All data were presented as means ± SD, and were declared significant at P < 0.05 according to Dytham (2011).

Results

Growth performance The growth and nutrients utilization of common carp, C. carpio, fed diets fortified with M. scaber leaves extract showed significant improvements when compared to the control group (P < 0.05; Table 2). Diets fortification with higher MSLE (10 g/kg diet) gave higher performance in terms of final weight, body weight gain, SGR, feed intake, and protein efficiency ratio, while least ones were recorded in fish fed the control diet. There were no significant difference was noticed in the fish survival (P > 0.05). Fish growth was significantly improved by dietary MSLE over that fed the control diet (P < 0.05).

10 Growth performance of Common carp fed MSLE-fortified diet

Table 2. Growth performance and nutrients utilization of Cyprinus carpio fed diets fortified with Mitrascarpus scaber extract for 84 days

MSLE levels (g/kg diets) Parameters 0.0 5.0 10.0 15.0 Initial weight (g) 7.52 ±0.23a 7.51 ±0.20a 7.52 ±0.14a 7.54 ±0.09a Final weight (g) 20.86 ±1.50b 22.61 ±2.03b 25.27 ±2.10a 25.70 ±1.33a Weight gain (g) 13.34 ±1.83c 15.10 ±1.70b 17.75 ±2.17a 18.16 ±1.15a Specific growth rate (%g/day) 1.21 ±0.03b 1.31 ±0.12b 1.44 ±0.22a 1.46 ±0.16a Feed intake (g) 32.13 ±0.12c 33.04 ±0.20b 33.13 ±0.05b 33.54 ±0.11a Feed Conversion Ratio 2.41 ±0.09a 2.18 ±0.08b 1.87 ±0.09c 1.85 ±0.05c Survival rate (%) 96.77 ±5.77a 95.00 ±8.66a 98.33 ±2.89a 98.33 ±2.89a Different superscripts in the same row are statistically significant different between means at P < 0.05.

Intestine morphometry The inclusion of MSLE in diets for common carp, C. carpio increased significantly villi length/width and absorption area of the intestine (P < 0.05). The increase in the intestine morphometry was observed with the increase in MSLE levels in diets; however, fish fed the basal diet had lowest villi length/width and area of absorption. Similarly, there was a progressive increase in the cryptal depth of the fish fed diets fortified with MSLE (P < 0.05; Table 3).

Table 3. Changes in morphometry of the intestine of Cyprinus carpio juveniles fed Mitrascarpus scaber extract based diets for 84 days

MSLE levels Villi length Villi width Cryptal depth Area of absorption (g/kg) (mm) (mm) (mm) (mm2) 0 0.32 ±0.03c 0.21 ±0.06c 0.14 ±0.01b 0.07 ±0.03c 5 0.54 ±0.08b 0.30 ±0.04b 0.19 ±0.04a 0.16 ±0.03b 10 0.63 ±0.02a 0.33 ±0.02b 0.21 ±0.01a 0.21 ±0.02a 15 0.64 ±0.20a 0.35 ±0.04a 0.24 ±0.03a 0.22 ±0.04a Different superscripts in the same column are statistically significant different between means at P < 0.05. Note: area of absorption (mm2) = Villi length (mm) × Villi width (mm).

Innate immune parameters The innate immune profile of fish fed diets supplemented with MSLE is shown in Table 4. The results indicated that supplementation of MSLE to common carp, C. carpio enhanced significantly the immunity parameters (P < 0.05). It was noticed that fish fed diet containing 10 g MSLE/kg diet had highest RBA (154.31 ±8.01), lysozyme activity (10.41 ±1.45), catalase (1.32 ±0.10 mg/g protein), and SOD (1.37 ±0.06 mg/g protein), and their least values were observe with fish fed the control diet (121.02 ±8.24, 9.53 ±1.20, 1.24 ±0.02 mg/g protein, and 0.43 ±0.01 mg/g protein, respectively). Furthermore, fish resistance against bacterial infection was improved. The post-challenge mortality of the fish infected withA. hydrophila is also shown in Table 4. There was a significant reduction in the post-challenge mortality (P < 0.05). Highest mortality (52.50 ±4.56%) was recorded in fish fed the control diet, while least one (8.45 ±1.30%) was observed in fish fed a diet containing 10 g MSLE/kg diet.

11 Ibrahim Adeshina, Benjamin O. Emikpe, Adetola Jenyo-Oni, Emmanuel K. Ajani, Musa I. Abubakar

Table 4. Changes in respiratory burst activity (RBA), lysozyme activity, Catalase, Superioxide dismutase (SOD) and post-challenge mortality of Cyprinus carpio juveniles fed Mitrascarpus scaber extract based diets for 84 days

MSLE levels RBA Lysozyme Catalase SOD Post-challenge (g/kg diet) (mg/mL) (U/mg protein) (mg/g protein) (mg/g protein) mortality (%) 0 121.02 ±8.24b 9.53 ±1.20a 1.24 ±0.02b 0.43 ±0.01c 52.50 ±4.56a 5 128.16 ±5.92b 9.58 ±1.11a 1.27 ±0.05a 0.84 ±0.03b 33.21 ±4.21b 10 143.29 ±6.11ab 10.27 ±0.98a 1.30 ±0.12a 1.32 ±0.01a 15.17 ±2.50c 15 154.31 ±8.01a 10.41 ±1.45a 1.32 ±0.10a 1.37 ±0.06a 8.45 ±1.30d Values are represented as mean ± standard deviation of triplicates; different superscripts in the same column are statistically significant different between means at P < 0.05.

Discussion

The inclusion of medicinal plants in fish diets has been studied, and positive results have been documented (Abdel-Tawwab, Ahmad, 2009; Abdel-Tawwab, Ahmad, Seden, Sakr, 2010; Abdel- Tawwab, 2018a; Abdel-Tawwab, 2012, 2015, 2016; Guardiola et al., 2016; Abdel-Tawwab, Abbass, 2017; Adeshina et al., 2017; Hoseinifar et al., 2017a, 2017b; Van Doan et al., 2017; Adeshina et al., 2018b). Their phytobiotics activity has made them suitable substitutes for antibiotics, growth promoters, and immune boosters. The present study, fish fed diets fortified with MSLE showed improved growth, and utilized the given diets efficiently when compared to the groups fed the basal diet. The higher growth observed herein might be associated with the higher feed consumed by fish fed MSLE-supplemented diets.The presence of antimicrobial, and antimycotic properties such as gallic acid, 3,4,5-trimethoxybenzoic acid, 4-Methoxyacetophenone, 3,4,5-trimethoxyacetophenone, n-octane, 2-Hexanol, p-Cymene, α and β pinene, eugenol etc might assist in reducing pathogenic organisms in fish gut; hence, promoting the colonization of beneficial bacteria that enhanced diets digestibility. The presence of the secondary metabolites MSLE may be stimulated its immunostimulant properties. The present of eugenol, and p-cymene in the plants could be responsible for it acceptability by the fish (Adeshina et al., 2018b). The results of the present study are in agreement with previous studies, which fed fish on phytobiotics additives (Abdel- Tawwab et al., 2018a; Sogbesan, Ahmed, Ajijola, 2017; Offor et al., 2014; Adewole, Faturoti, 2017; Harikrishnan, Balasundaram, Heo, 2011). Other studies (Adeshina et al., 2018b; Abdel-Tawwab et al., 2018a; Sogbesan et al., 2017) reported that fish fed varying inclusion levelsof plants extracts had higher growth performance that the growth fed the basal diet. The present study revealed that the increase in fish weight with the increase in the MSLE levels in diets more than the control diet. Thus, such increase in the body weight of the fish fed MSLE-based diets suggests better ingestion, digestion activity, and nutrient absorption. One of the tools that researchers have adopted in assessing the nutrients absorption in fish is the examination of gut morphometry. The improvements observed in villi length/width, cryptal depth, and area of absorption of common carp, C. carpio juveniles fed MSLE-based diets revealed the improvements of nutrients utilization by fish. These increases in gut morphometry in the present study supported the increase in fish weights better than observation in the control group especially fish fed 10 g MSLE/kg diet. These results are in concomitant with previous studies (Zhou et al., 2010; Zahran, Risha, AbdelHamid, Mahgoub, Ibrahim, 2014; Zhang et al., 2010). Furthermore, it has been reported that inducement of intestinal morphology increases nutrient

12 Growth performance of Common carp fed MSLE-fortified diet utilization ability of the fish leading to increasing fish growth, and feed utilization (Dimitroglou et al., 2010; Zhou, Buentello, Gatlin, 2010; Zahran et al., 2014). In similar studies, Abdel-Tawwab et al. (2018a) and Adeshina et al. (2018b) found significant increases in villi length/width and areas of absorption of African catfish,Clarias gariepinus fed diets fortified with plants extracts. The fish survival in all treatments was higher than 95% which validated that MSLE has no toxic effect on fish. This result is in agreement with the earlier findings revealing the successful usage of aromatic plants in fish diets (Ainsworth, 1992; Saeidi, Adel, Caipang, Dawood, 2017; Awad, Austin, 2010; Abdel-Tawwab, Sharafeldin, Mosaad, Ismaiel, 2015; Brum et al., 2017; Abdel-Tawwab et al., 2018b). The innate immune response of common carp, C. carpio fed MSLE-enriched diets improved significantly (P < 0.05) better than fish fed the basal diet. Higher RBA, lysozyme, SOD, and catalase activities may be attributed to the bioactive ingredients of MSLE. These active ingredients perform chemopreventive, free radical scavenging, and antimycotic activities (Adeshian et al., 2018a; Abdel-Tawwab et al., 2018a). Also, several phenolic compounds with antioxidant properties are presence in MSLE. Reports have shown that RBA and lysozyme activities are important immune parameters, which play a significant role as defense mechanism against many invading agents (Adel et al., 2015; Jayashree, Subramanyam, 1999). As revealed in the present study, MSLE stimulates respiratory burst, and lysozyme activities. Moreover, dietary MSLE enhanced the fish resistance against A. hydrophila infection. Abdel-Tawwab et al. (2018a) reported that disease challenge trial is a critical way to investigate the effectiveness of phytobiotics on the immune system of fish. The reduction in post-challenged fish mortality observed in the present study is associated with immunomodulatory effectof MSLE. These results may be because this plant has antibacterial and antifungal activities (Bisignano et al., 2000; Abere et al., 2007; Anejionu et al., 2011). Furthermore, usefulness of MSLE active ingredients is their hydrophobicity and phagocytic activities (Chou, Hung, Lin, Lee, Leu, 2010; Zahran et al., 2014). Several studies indicated that feed supplemented with various medicinal plants improved their resistance against pathogens bacteria (Adeshian et al., 2018a; Abdel-Tawwab, 2012, 2015, 2016; Abdel-Tawwab, Abbass, 2017; Abdel-Tawwab, Ahmad, 2009; Abdel-Tawwab et al., 2010; Guardiola et al., 2016; Van Doan et al., 2017; Hoseinifar et al., 2017a, 2017b; Adeshina et al., 2017; Abdel-Tawwab et al., 2018a; Owolabi et al., 2014; Irobi, Daramola, 1993).

Conclusions

Growth performance and nutrient utilization of common carp fed MSLE-fortified diets were significantly improved over the control diet with optimum levelof 10 g MSLE/kg diet. The present study proffers a new outlook for the use of M. scaber as phytobiotics to boost fish growth, and immunity. Furthermore, MSLE inclusion in the diet of common carp, C. carpio, could be used to enhance intestinal morphology, and resistance against bacterial infection with A. hydrophila.

Ethical statement The protocol of the study was subjected to ethical consideration and was approved by Animal Care Use and Research Ethics Committee, University of Ibadan, Ibadan, Oyo state, Nigeria with reference number UI-ACUREC/App/03/2017/008.

13 Ibrahim Adeshina, Benjamin O. Emikpe, Adetola Jenyo-Oni, Emmanuel K. Ajani, Musa I. Abubakar

Conflict of interest We declared that there are no conflict of interests.

References

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Cite as: Adeshina, I., Emikpe, B.O., Jenyo-Oni, A., Ajani, E.K., Abubakar, M.I. (2019). Growth performance, gut morphometry and innate immune profiles of common carp, Cyprinus carpio juveniles fed diet fortified with Mitracarpus scaber leaves extract and its susceptibility to pathogenic bacteria, Aeromonas hydrophila. Acta Biologica, 26, 5–17. DOI: 10.18276/ab.2019.26-01. #1#

#0#

Acta Biologica 26/2019 | www.wnus.edu.pl/ab | DOI: 10.18276/ab.2019.26-02 | strony 19–30

Histopathology of the gills, livers and kidney of Clarias gariepinus (Burchell, 1822) exposed to sniper 1000EC under laboratory conditions

Musa I. Abubakar,1 Ibrahim Adeshina,2 Ikililu Abdulraheem,3 Somira Abdulsalami4

1 Department of Aquaculture and Fisheries, Faculty of Agriculture, University of Ilorin, P.M.B. 1515 Ilorin, Kwara State, Nigeria 2 Department of Aquaculture and Fisheries, Faculty of Agriculture, University of Ilorin, P.M.B. 1515 Ilorin, Kwara State, Nigeria 3 Department of Aquaculture and Fisheries Management, Faculty of Agriculture, federal university of Agriculture, P.M.B. 1313 Abeokuta, Ogun State, Nigeria 4 Department of Biological Sciences, Fisheries and Aquaculture, College of Natural and Applied Sciences, Cresent University, P.M.B 2104, Abeokuta, Ogun State, Nigeria

Corresponding authors, email: [email protected], [email protected], [email protected], [email protected]

Keywords Sniper 1000EC, histopathology, Clarias gariepinus, gills, livers and kidney Abstract Indiscriminate use of Sniper 1000EC has become a serious problem among local fishermen in the Northern parts of Niger state. Juveniles of Clarias gariepinus (mean body weight 23.34 ±0.05 g; mean standard length, 20.00 ±0.45 cm) were subjected to 5 treatment levels of 0, 5, 10, 15 and 20 mg/l. The tissue damages were observed majorly at 15 and 20 mg/l of sniper 1000EC exposed fish species. Oedema and hyperplasia were observed in the gills of the exposed groups. Hepatocellular steatosis and vacuolations were observed in the livers. Tubular nephrosis and hyperplasias of epithelial cells were also observed in the kidney. It is concluded that alterations in gills, livers and kidney of the exposed fish species were consequences of exposure to the toxicant (sniper 1000EC). It is recommended that the use of Sniper 1000EC by local fishermen be banned to save the aquatic environment from destruction.

Obraz histopatologiczny skrzeli, wątroby i nerek suma afrykańskiego Clarias gariepinus (Burchell, 1822) narażonego na środek owadobójczy Sniper 1000EC w warunkach laboratoryjnych. Słowa kluczowe Sniper 1000EC, histopatologia, Clarias gariepinus, skrzela, wątroba i nerki Streszczenie Bezkrytyczne stosowanie Sniper 1000EC stało się poważnym problemem dla lokalnych rybaków w północnych częściach Nigru. Młode osobniki Clarias gariepinus (średnia masa ciała 23,34 ±0,05 g; średnia długość 20,00 ±0,45 cm) przetrzymywano w warunkach laboratoryjnych w środowisku zawierającym 0, 5, 10, 15 i 20 mg/l środka Sniper 1000EC. Uszkodzenia tkanek obserwowano głównie przy najwyższych stężeniach (15 i 20 mg/l). Obserwowano obrzęk i przerost skrzeli narażonych ryb. W wątrobie obserwowano stłuszczenie i wakuolację.

19 Musa I. Abubakar, Ibrahim Adeshina, Ikililu Abdulraheem, Somira Abdulsalami

W nerkach stwierdzono nefrozę i przerost komórek nabłonkowych. Wykazano, że zmiany w skrzelach, wątrobach i nerkach narażonych ryb były konsekwencjami narażenia na działanie insektycydu (Snajper 1000EC). Ze względu na dużą toksyczność zaleca się zakazać używania Sniper 1000EC.

Introduction

Histopathological changes have been widely used as biomarkers in the evaluation of the health of fish exposed to contaminants in laboratory animal (Wester, Canton, 1991; Thophon et al., 2003; Abubakar, Adeshina, 2019) and wild specimen (Hinton et al., 1992; Schwaiger et al., 1997; Tech, Adams, Hinton, 1997; Drishya, Binu-Kumari, Mohan-Kumar, Ambikadevi, Aswin, 2016). One of the great advantages of using histopathological biomarkers in environmental studies is that this category of biomarkers allows the examination of the target organs (Gernhofer, Pawet, Schramm, Muller, Triebskorn, 2001). Furthermore, the alterations found in these organs are clearer than functional ones (Fanta, Rios, Romao, Vaaianna, Freiberger, 2003). It also reveals signs of damage to animal health (Hinton & Lauren 1990). The gills, being a respiratory organ of fish (Banerjee, 2007) is frequently in contact with external environment and thus vulnerable to aquatic toxicants (Abubakar, 2013). Studies have been conducted on histopathological changes in the gills, liver and kidney of fish exposed to various substances (Auta, 2001; Drishyaet al., 2016; Abubakar, Adeshina, 2019) including pesticide which have been reported to cause pathological alteration in the exposed C. gariepinus (Auta, 2001). The liver ow f fish can be considered as a target organ to pollutants, alterations in its structure can be significant in the evaluation of fish health (Kolbasi, Burcu, Ucuncu, Onen, 2009). Widespread application of various pesticides has aggravated the problem of pollution to aquatic environment. Due to these synthetic chemicals, environment has failed to keep its healthy characteristics. The insecticides of proven economic potentialities could not do well in the ecosystem when viewed on extra fronts since these revenue poisons, in a residual form or as a whole, get into the aquatic ecosystem. They cause a series of problems to aquatic organisms (Camargo, Martinez, 2007; Mastan, Ramayya, 2010; Drishya et al., 2016; Abubakar, Adeshina, 2019). Sniper 1000EC (2, 3-dichlorovinyl dimethyl phosphate), a brand of dichlorvos, is contact acting and fumigant insecticide (Abubakar, 2013). Like all organophosphates, it kills insects and other target organisms because of its toxicity to the nervous system. This is achieved by inhibition of enzyme acetylcholinesterase (AchE) that breaks down acetycholine at the receptor site for partial uptake into the nerve terminal (Drishya et al., 2016). Without functioning AchE, accumulation of acetylcholine results in depolarizing block of muscle membrane, producing rapid twitching of involuntary muscles, convulsions, paralysis and early death (Camargo, Martinez, 2007; Mastan, Ramayya, 2010; Drishya et al., 2016; Abubakar, Adeshina, 2019). Indiscriminate use of Sniper 1000EC is common among local fishermen from Northern partsof Niger state. The African catfish,Clarias gariepinus is an important food fish in Nigeria (Abubakar, 2012). Clarias gariepinus is not only the most predominant fish species raised in aquaculture in Nigeria, but has also served as an experimental model of aquatic vertebrate for two decades (Cavaco et al., 2001). Despite the indiscriminate use of Sniper 1000EC by local fishermen, there is a paucity of information on its toxicity. The aim of the present study was to evaluate histological alterations in gills, liver and kidney of Clariasgariepinus (Burchell, 1822) exposed to sniper 1000EC under laboratory conditions.

20 Histopathology of the fish tissues exposed to sniper 1000EC

Materials and Methods

Experimental fish and test chemical Juveniles of Clarias gariepinus (n 150, mean body weight 23.34 ±0.05 g) were purchased from a reputable 36 fish farms in Ilorin, Kwara State, Nigeria. The samples were transported to the Central Laboratory, University of Ilorin in plastic container of 100 L capacity filled with water to two-third volume between 07:00 hours and 09:00 hours. They were held in large water baths of 160 L capacity and acclimated for 14 days to laboratory conditions. The top of water bath was covered with netted material to prevent jumping out of the fish. A slit was made at middle of the net to allow for feeding fish and cleaning of the bath. Feeding commenced two days after the arrival and stopped twenty-four hours before the commencement of the experiment. During acclimation, fish were fed twice daily (08:00 and 16:00 hours) with formulated feed (45% crude protein, Table 1) at 5% body weight.

Table 1. Proximate composition of experimental diets

Parameters Composition (%) Moisture 8.3 Crude protein 45.0 Ash 9.5 Crude lipid 12.0 Crude fibre 1.5

The fishwere accepted and adapted to laboratory conditions for the 14 days.The water in the bath was changed daily and uneaten food and faecal matters were siphoned out. Water parameters were monitored two times daily at the hours of 08:00 and 18:00. The water temperature, DO and pH ranges throughout the experimental period were 24.1–25.7°C, 5.6–6.6 mg/l, and 7.24–7.81, respectively which were measured with the aid of mercury-in-glass thermometer, a digital DO meter (Model AVI-660, Labtech International Ltd, Heathfield, UK) and a digital pH-meter (Model Photoic 20, Labtech International Ltd, Heathfield, UK), respectively. Dead fish were also removed to minimize contamination of water. Test chemical (2, 3-dichlorovinyl dimethyl phosphate), a brand of Dichlorvos with the trade name Sniper 1000EC (United Phosphorous Limited, UPL) was obtained from Minna central market and was used for the study. The test concentrations were prepared with reference to the Manual of Method in Aquatic Environment Research.

Experimental design The experimental design was a complete randomized design. A total of one hundred and fifty (150) juvenile of Clarias gariepinus were randomly distributed into the tanks (60 × 38 × 27 cm) at a stocking rate of 10 fish per tank.The fifteen (15) tanks were assigned to 5 treatments comprises 0, 5, 10, 15 and 20 mg/l. Dead fish from experimental groups were removed and dissected immediately for histological analysis while specimens from control were vivisected without anesthesia and subjected to histological analysis at the end of one- week experiment.

21 Musa I. Abubakar, Ibrahim Adeshina, Ikililu Abdulraheem, Somira Abdulsalami

Histological procedures Samples of fish from both control and experimental groups were excised, rinsed in physiological saline and fixed in Bouin’s fluid for 6, 12 and 8 hours respectively. The gills, livers and kidneys were subjected to micro techniques (Bucke, 1989; Adeshina, Jenyo-Oni, Emikpe, Ajani, Abdel-Tawwab, 2019). The tissues were dehydrated in an ethyl alcohol series of ascending concentrations, embedded in paraffin and sectioned at 5 µm thickness.The tissues sectioned were stained with haematoxilin-eosin (HE). Stained slides were cleared in xylene and mounted in synthentic resin medium. The slides were examined under low power (×4) objective and high power (×10) objective with Tension Binocular microscope. Under extreme low power, the condenser was moved into a very low position to prevent the image of the lamp bulb from obscuring the image of the micrographs. Photomicrographs were then taken at low power (×40) objective with Samsung ES25 4X Zoom Lens digital Camera and downloaded into a computer. Photomicrographs of control groups were compared with those of exposed groups under the guidance of a pathologist.

Results

Histopathology of the gill No alterations were observed in the gills of the control (Figure 1A). The most common pathological changes in the gills of exposed fish species were fusion of lamellae, oedema and hyperplasia of the secondary lamellae (Figure 1B).

A B Showing normal appearance of the gill lamellae (A) and Photomicrograph of sections of the gill of Clarias gariepinus exposed to 20 mg/l of sniper 1000EC (B) showing fusion of lamellae, oedema and hyperplasia of interlamellae Figure 1. Photomicrograph of a section of the gill from a control Clarias gariepinus

Histopathology of the liver There were no abnormalities in liver of the control groups (Figure 2A .The pathologicalchanges in the liver tissues of the exposed fish species showed distortions with steatosis,vacuolations and necrosis. (Figure 2B).

22 Histopathology of the fish tissues exposed to sniper 1000EC

A B Showing normal appearance (A) and Photomicrographs of sections of the liver of Clarias gariepinus exposed to 15 mg/l of sniper 1000EC (B) showing steatosis vacuolation and necrosis . Figure 2. Photomicrograph of a section of the liver from a control Clarias gariepinus

Histopathology of the kidney There were no observed distortions in the kidneys of the control group (Figure 3A).Exposed kidneys had marked cellularity of tubules infiltrated by lymphocytesand neutrophils. The interstitial were heavily infiltrated by inflammatory cells.The tubular cells were hypertrophic and the lumina contained armorphouseosinophilic materials (Figure 3B).

A B Showing homogeneous parenchyma tissues (A) and photomicrograph of sections of the kidney of Clarias gariepinus exposed to 20mg/l of sniper 1000EC (B) showing tubular nephrosis and hyperplasia. Figure 3. Photomicrograph of section of anterior kidney from a control Clarias gariepinus

Discussion

Gills are the primary corridor for molecular exchange between the internal milieu of fish and their external environment (Olson, 1996), such as gas transfer, acid-base regulation and ionic regulation (Eddy, 1982). The filamentof gills and their secondary lamellae represent two general types of epithelium (Laurent, Dunel, 1980; Laurent, 1984) that contain three cell epithelia, the pavement, the chloride (ionocyce or mithochondria- rich) cells and the mucous cells, which are most prevalent. In some fish species, the gills contain microvillous or smooth surface lamellar

23 Musa I. Abubakar, Ibrahim Adeshina, Ikililu Abdulraheem, Somira Abdulsalami pavement cells (Hossler, Harpole, King, 1986). Study of the gills in the control fish showed a typical structural organization of the lamella while gills from the fish exposed to the toxicant had several histological distortions, namely clumping/fusion, oedema and hyperplasia of the lamellae. The gill abnormalities observed in this study were similar to previous studies on fish gill morphology, which showed separation of the epithelial layers of secondary gill lamellae, deformation of secondary lamellae and degeneration of chloride cells accompanied by hyperplasia of undifferentiated cells in the primary lamellae (Daye, Garside, 1976; Chevailer, Gauthier, Moreau, 1985). The oedema and hyperplasia in the gills of the exposed fish might be due to acute inflammatory nature of the lesion induced in such gills by the sniper 1000EC. Eller (1970) reported hyperplasia of gill lamella epithelium as findings suggestive of toxicity in several fish species following exposure to several toxicants. Oedema with epithelial separation was reported by (Eller, 1971; Van Valin, Andrews, Eller, 1968) as additional gill change in fish exposed to toxicants. Epithelia sodium pump failure might be responsible for gill oedema (Mitchel, Cotran, 2004). The hyperplasia might in addition be an attempt to increase blood supply to compromised gill in order to increase blood oxygenation. (Gabriel, Amakiri, Ezeri, 2007). This could be a com- pensatory maechanism in the exposed fish to counter hypoxia sequel to edema and obliterated inter-lamellae space, which impeded gaseous exchange across such gills (Gabriel et al., 2007). Oedema has been reported to increase the diffusion distance between gill capillaries and epithelial cells, thereby increasing internal diffusion resistance, which is a major factor that inhibits gaseous exchange across the affected gills (Turala, 1983). The lamellar fusions are defense mechanisms that reduce the branchial superficial area in contact with the toxicant. These mechanisms also increase the diffusion barrier to the pollutant (Lauren, Mc Donald, 1985; Van Heerden, Vosloo, Nikinmaa, 2004). Similarly, fusion of adjacent secondary lamellae as a consequence of edema could lead to telangiectasis, which is a characteristic pathologic feature of compromised gills associated with physical or chemical damage (Robert, 2001). However, such structural changes are reported to be non-toxicant specific (Meyer, Hendricks, 1985), which could be mere stress response of fish, to toxicants exposures (Gabriel et al., 2007). The effect of sniper 1000EC that brought about edema, hyperplasia and laceration of gill lamella explains why fishermen used sniper 1000EC indiscriminately causing heavy fish kills as it destroys the gills, the major respiratory and food filtering organ in fish (Abubakar, 2013). It was recognized that liver is difficult to analyse due to its high histological variability (Kotin, 1967). However, Myers, Landahi, Krahn, Johnson, McCain (1991) and Myers, Olson, Johnson, Stehr, Varansi (1992) have described the effectsof different toxicants on the liversof various fish species. They showed that the liver distortions developed gradually over the time of exposure and that this varies from individual fishes. They also showed that it is possible that liver could regenerate and recover from those distortions. In this study, the histopathologies of the liver of C. gariepinus were carried out with a view to explaining the toxic mechanism of sniper 1000EC. The toxicant caused alterations of the liver parenchyma, such as vacuolization and necrosis. These alterations were often associated with a degenerative- necrotic condition (Myers, Rhodes, McCain, 1987). This is in agreement with the reports of Omoregie, Ufodike (1990) and Auta (2001) who separately reported reduced liver vacoulation in Oreochromisniloticus exposed to actellic 25EC, paraquat and dimethoate respectively.The reduced vacoulation of hepatocytes may be due to fatty degeneration (Thiyagarajah, Grizzle, 1985; Auta, 2001; Langiano, Martinez, 2008). Vacuolation of hepatocytes with pycknotic (condensed) nuclei in the liver was most likely due to deposition of glycogen and lipid (Myers et al., 1987) as a result of hepatotoxicity induced by

24 Histopathology of the fish tissues exposed to sniper 1000EC the presence of the toxicant and/or reduced food intake (Khan, Kiceniuk, 1988). Ferguson (1989) observed that a common morphologic response of fish liver to toxicity is the loss of hepatic glycogen and/or lipid. This condition according to Wolfe and Wolfe (2005) might have occurred by direct intoxication or it might have occurred secondarily to deceased body condition caused by stress or concurrent disease. Furthermore, the authors noted paradoxically that toxic exposure might also result in the accumulation of fats as recorded in this study. The steatosis and necrosis were similar to those reported for fish caught in contaminated water or those exposed to various chemicals under laboratory conditions (Brand, Fink, Bengeyfield, Birtwel, Mc Allister, 2001; Rudolph, Yanez, Troncoso, 2001; Marty, Hoffinamn, Okihiro, Hepler, Hanes, 2003; Fafioye, Adebisi, Fagade, 2004; Koehler, 2004; Olojo, Olurin, Mbaka, Oluwemimo, 2005; Camargo, Martinez, 2007; Wahbi, El-Greishy, 2007; Aniladevi, Philip, Smitha, Bhanu, Jose, 2008). Fatty degeneration of the liver (haemosiderosis/steatosis) recorded in the liver of the exposed fish might be suggestive of metabolic disorders and it was commonly associated with dietary deficiency in response to xenobiotic (Myers et al., 1987). These changes are normally reported in disease organisms or those exposed to toxicants (Khan, Kiceniuk, 1988; Hawkins, Walleewrr, Orerstreet, Lyte, Lyte, 1988; Ogbulie, Okpkwasili, 1999). It might have resulted from disturbances in any of the steps in the sequence of the events from fatty acid entry to lipoprotein exit (Mitchell, Cotran, 2004). The degree of fatty changes in the exposed species exceeded that of the control implicating the toxicant as being responsible for the change. Lipid or glycogen vacuolation suggested the accumulation of triglycerides usually within the hepatocytes and may be responsible for the hepatocyte enlargement/hyalination (Wolf, Wolf, 2005). Steatosis has been correlated with neoplasms; however, its role in the progression of lesions towards neoplasm formation in fish was not well understood (McCain et al., 1982). Similar changes have been reported in the liver of Astyanax sp exposed to WSFs of crude oil (Akaishi et al., 2004). Percafluviatilis and gold fish exposed to oil seed process affected water (Neroet al., 2006), centrolobular necrotic change was the main change in the species exposed to the toxicant and agrees with the observation of Popp (1991) who suggested that the distribution of the interstitial system in the liver resulting in a higher concentration of the toxicant in the centrolobular region account for the occurrence and frequency of centrolobular toxicity. The necrosis of the liver tissue might be due to inability of the fish to regenerate new liver cells due to the effectof sniper 1000EC. Several studies have reported that chronic accumulation of some toxicant in fish livers causes hepatocyte lysis, cirrhosis and eventually death (Pourahamad, O’Brien, 2000; Varanka, Rojik, Varanka, Nemcsók, Ábrahám, 2001). The kidney has been established as one of the principal site of erythropoietin production (Gordon, Zanjani, 1970). As in higher vertebrates, the kidneys of fish perform an important function related to electrolyte and water balance and maintenance of a stable internal environment. Thurston, Russo and Smith (1978) reported mild hydrophobic degeneration in renal tubule. The hydrophobic degeneration observed in the kidneys of the exposed fish species might be due to an increase in the perme- ability of fish tissues to water, and increased urine out put as reported by Lloyd and Orr (1969) for rainbow trout. Following the exposure of the fish to the toxicant, histological alterations have been found at the level of tubular epithelium (Teh et al., 1997). Ortiz, de Canales and Sarasquete (2003) found kidneys of exposed fish to have received the largest proportion of post-branchial blood, and therefore renal lesions may be good indicators of environmental pollution. Any effects on the kidney are likely to lead to major problems of anaemia and a lack of new blood cells formation. In this study, there was marked diffused tubular necrosis, hyperplasia of interstitial haemopoietic tissues.

25 Musa I. Abubakar, Ibrahim Adeshina, Ikililu Abdulraheem, Somira Abdulsalami

The changes recorded in the kidney section from exposed fish were similar to those caused by petroleum in English sole (McCain et al., 1978) and rats (Dede, Kagbo, 2001) and C.gariepinus exposed to plant extracts (Onusiruka, Ufodike, 2000; Fafioye et al., 2004) for various periods. Peripheral blood and cephalic kidney of turbot, Scophthalmusmaximus and Atlantic cod, Gadusmorus had micronuclei and severe nuclei abnormality such as nuclear buds, binucleated and nonylphenol (Barsiene, Dedonvte, Rybakovas, Anderson, 2006). Exposure, such as changes in kidney tubules particularly at the highest concentration recorded in this study might greatly impair the infiltration functions of the kidney with grave consequences for the exposed fish. However, the onset development and extent of these changes might be toxicant concentration and exposure time dependent.

Conclusion

In this study, alterations in gills, livers and kidney in the exposed fish species were associated with the effects of different concentrations of sniper 1000EC with LD50 of 15 mg/l. By this context, smipper at 15 mg/l causes significant damage to fish tissue and hence be prevented and prohibited for fishing.

References

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28 Histopathology of the fish tissues exposed to sniper 1000EC

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29 Musa I. Abubakar, Ibrahim Adeshina, Ikililu Abdulraheem, Somira Abdulsalami

Van Heerden, D., Vosloo, A., Nikinmaa, M. (2004). Effects of short-term copper exposure on gill structure, me thallothionein and hypoxia-inducible factor-1. (HIF-1-) levels in rainbow trout (Oncorhyynchus mykiss). Aquatic Toxicology, 69, 271–280. Van Valin, C.C., Andrews, A.K., Eller, L.L. (1968). Some effects of mirex on twowarm-water sites. Transi- tion American Fisheries Society, 97–185. Varanka, Z., Rojik, I., Varanka, I., Nemcsók, J., Ábrahám, M. (2001). Biochemical and Morphological changes in carp (Cyprinus carpio L.) liver following exposure to copper sulfate and tannic acid. Com- parative Biochemistry and Physiology, 128, 467–478. Wahbi, O.M., El-Greisy, Z.A. (2007). Comperative impact of different waste sources onthe the reproductive parameters and histology of gonads, liver and pituitary gland of Siganus rivaltus. Journal of Applied Science Research, 3, 236–244. Wester, P.W., Canton, J.H. (1991). The usefulness of histopathalogy in aquatictoxicity studies. Comparative Biochemistry and Physiology, 100, 115–117. Wolfe, J.C., Wolfe, J.W. (2005). A brief review of non neoplastic hepatic toxicityin fish. Toxicology and Pathology, 33, 75–83.

Cite as: Abubakar, M.I., Adeshina, I., Abdulraheem, I., Abdulsalami, S. (2019). Histopathology of the gills, livers and kidney of Clarias gariepinus (Burchell, 1822) exposed to sniper 1000EC under laboratory conditions. Acta Biologica, 26, 19–30. DOI: 10.18276/ab.2019.26-02. #1#

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Acta Biologica 26/2019 | www.wnus.edu.pl/ab | DOI: 10.18276/ab.2019.26-03 | strony 31–34

First record of Thecacineta calix (Ciliophora: Suctoria) on harpacticoid copepod from Aegean Sea, Turkey

Furkan Durucan

Işıklar Caddesi No. 16, TR-07100 Antalya, Turkey

Corresponding author: email: [email protected]

Keywords Suctorians, epibiosis, environment, host specificity, diagnosis Abstract The present study report a marine suctorian species Thecacineta calix (Schroder, 1907) on harpacticoid copepod collected from Fethiye (Muğla), Aegean Sea, Turkey. This is the first record of this suctorian ciliates from Turkey. It is also first recordof this species from Aegean Sea.

Pierwsze stwierdzenie Thecacineta calix (Ciliophora: Suctoria) widłonoga z rzędu Harpacticoida z Morza Egejskiego, Turcja Słowa kluczowe sysydlaczki, epibioza, środowisko, wybiórczość gospodarza, diagnoza Streszczenie W artykule zaprezentowano pierwsze stwierdzenie w Turcji i na Morzu Egejskim morskiego sysydlaczka Thecacineta calix (Schroder, 1907) stwierdzonego w Fethiye (Muğla), Morze Egejskie, Turcja.

Introduction

Suctorian ciliates can be found in all types of water bodies on a wide diversity of hosts and substrates. They inhabit both anorganic and organic material, plants and aninals and feed micro- algae and other ciliates. Several suctorian ciliates are common epibionts of benthic marine and interstitial invertebrates. Marine and fresh water mites have been identified as hostsof suctorian ciliates which may be commensals, ecto – or endoparasites. They are common epibionts on marine and freshwater invertebrates such as copepods, cladocerans, nematodes, kinorhynchs, halacarid and hydrachnid mites (Chatterjee, Nanajkar, Dovgal, Sergeeva, Bhave, 2019; Durucan, Boyacı, 2016; Durucan, Artüz, Dovgal, 2019). In Turkey, the first taxonomic study on marine suctorians was done by Durucan and Boyacı (2019) who reported Praethecacineta halacari (Schulz, 1933) on Copidognathus venustus Bartsch, 1977 from Levantine Sea (Antalya). After that, Durucan et al. (2019) reported Paracineta irregularis Dons, 1928 on Rhombognathus sp. from Sea of Marmara.

31 Furkan Durucan

In the present study, specimens of Thecacineta calix were observed on a marine harpacticoid copepod as epibionts for the first time in Turkey. This record also new for Aegean Sea.

Material and Methods

The present study is based on material collected by the author whilst studying halacarids from Fethiye, Muğla (September, 2019) (36.602517°N, 29.030953°E) (Figure 1). Sediment samples were collected by using SCUBA at locality. After that sediment samples were sieved in 100 µm in the laboratory under a binocular microscope (Nikon SMZ 10). Drawings were made using a camera lucida microscope (Nikon Eclipse E400). The light microscopy photographs were taken with camera phone (Honor Play 8A). The ciliated harpacticoid copepod specimen mounted in Hoyer’s medium and kept in the author’s personal collection in Antalya.

Figure 1. Map of the study area (black square) showing the sampling station (Fethiye, Muğla)

Results and Discussion

Systematics Class Suctorea Claparede & Lachmann, 1859 Subclass Vermigenia Jankowski, 1978 Order Spelaeophryida Jankowski, 1978 Family Thecacinetidae Matthes, 1956 Genus Thecacineta Collin, 1909 Thecacineta calix (Schroder, 1907) (Figure 2)

Material examined Five suctorian ciliates identified asThecacineta calix have been observed on the cuticle of the harpacticoid copepod collected from medium coarse sand, at a depth of 10 m, in Fethiye, Muğla (September, 2019) (36.602517°N, 29.030953°E) (Figure 1). Coll. F. Durucan.

32 First record of Thecacineta calix from Turkey

Diagnosis Marine loricate suctorian. Whole lorica cuticle with covered by annular ridges (13–16). Length of lorica 100–110 μm; width of lorica 50–60 μm. Macronucleus large, oviform, located at the bottom of the cell body (Dovgal, Chatterjee, Ingole, 2008) (Figure 2).

Figure 2. Posterior part of harpacticoid copeod with Thecacineta calix marked with an asterisk

Distribution and host specificity

In this study, only a ciliated harpacticoid copepod has been found from the sampling area while studying on halacarid mites along Fethiye coast. Thecacineta calix (Schroder, 1907) was described by Schroder (1907) from a marine nematodes from Kerguelen Islands, Antarctica. The species has distributed worldwide (Chatterjee et al., 2019). However, the species was recorded only from the Adriatic Sea (Rovinj and Trieste) in the Mediterranean basin (Chatterjee et al., 2019). Including this study, total number of known marine suctorian ciliate is reach 2 to 3 in Turkey after reporting Paracineta irregularis Dons, 1928 and Praethecacineta halacari (Schulz, 1933) (Durucan, Boyacı, 2019; Durucan et. al., 2019).

Acknowledgements I would like to thank due to Isparta University of Applied Sciences, Eğirdir Fisheries Faculty, Biology, Ecology and Limnology laboratory (Isparta, Turkey) for providing laboratory facili- ties. I am also very thankful to anonymous reviewers for their constructive comments on the manuscript.

33 Furkan Durucan

References

Chatterjee, T., Nanajkar, M., Dovgal, I., Sergeeva, N., Bhave, S. (2019). New records of epibiont Theca- cineta calix (Ciliophora: Suctorea) from the Caspian Sea and Angriya Bank, Arabian Sea. Cahiers de Biologie Marine, 60, 445–451. Dovgal, I.V., Chatterjee, T., Ingole, B. (2008). An overview of Suctorian ciliates (Ciliophora, Suctorea) as epibionts of halacarid mites (Acari: Halacaridae). Zootaxa, 1810, 60–68. Durucan, F., Boyacı, Y.Ö. (2016). First Record of the Ciliate Praethecacineta halacari (Ciliophora: Suc- torea) Epibiont on Copidognathus Halacarid Mite from Portugal. Süleyman Demirel Üniversitesi Eğirdir Su Ürünleri Fakültesi Dergisi, 12 (2), 97–100. Durucan, F., Artüz, M.L., Dovgal, I.V. (2019). The first record of Paracineta irregularis (Ciliophora, Suc- torea) as epibiont on Rhombognathus halacarid mite (Acari, Halacaridae) from the Sea of Marmara, Turkey. Protistology, 13 (2), 67–70. Durucan, F., Boyacı, Y.Ö. (2019). First record of Praethecacineta halacari (Suctorea: Ciliophora) from Antalya, Turkey. Acta Aquatica Turcica, 15 (2), 135–138. DOI: 10.22392/actaquatr.577448.

Cite as: Durucan, F. (2019). First record of Thecacineta calix (Ciliophora: Suctoria) on harpacticoid copepod from Aegean Sea, Turkey. Acta Biologica, 26, 31–34. DOI: 10.18276/ab.2019.26-03. #1#

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Acta Biologica 26/2019 | www.wnus.edu.pl/ab | DOI: 10.18276/ab.2019.26-04 | strony 35–44

Biofilm and Quorum Sensing inArchaea

Małgorzata Pawlikowska-Warych,1 Beata Tokarz-Deptuła,2 Paulina Czupryńska,3 Wiesław Deptuła4

1 Department of Microbiology, Faculty of Biology, University of Szczecin, Felczaka 3c, 71-412 Szczecin, Poland 2 Department of Immunology, Faculty of Biology, University of Szczecin, Felczaka 3c, 71-412 Szczecin, Poland 3 Department of Microbiology, Faculty of Biology, University of Szczecin, Felczaka 3c, 71-412 Szczecin, Poland 4 Nicolaus Copernicus University in Toruń, Faculty of Biological and Veterinary Sciences, Institute of Veterinary Medicine, Gagarina 7, 87-100 Toruń, Poland

Corresponding author, e-mail: [email protected]

Keywords Archaea, biofilm, environment, quorum sensing Abstract In the article, we presented a brief description of the Archaea, considering their structure, physiology and systematics. Based on the analysis of the literature, we bring closer the mechanism of biofilm formation, including extracellular polymeric substances as well as cellular organelles, such as archaella, pili and ‘hami’. The method of forming a biofilm depends on the type of Archaea and the environment in which it naturally lives. We are also introducing the phenomenon of quorum-sensing, as a mechanism of communication of Archaea in the environment. This phenomenon corresponds to similar molecules as bacteria, namely acylated homoserines lactones, QS peptides, autoinducer-2 and -3 and others. In the case of biofilms and the occurrence of the phenomenon of quorum sensing, it can be concluded that these phenomena are very important for the life of Archaea. The phenomena described seem to be conservative, because both in Archaea and Bacteria are regulated by the same mechanisms.

Biofilm i zjawisko “quorum sensing” u Archaea Słowa kluczowe Archea, biofilm, środowisko, quorum sensing Streszczenie W artykule przedstawiamy opis Archaea, z uwzględnieniem ich budowy, fizjologii i systema- tyki. Na podstawie piśmiennictwa przybliżamy mechanizm tworzenia biofilmu wraz z poza- komórkowymi substancjami polimerowymi, a także typowe tylko dla archeonów organelle komórkowe, takie jak archaella, pili i „hami”. Metoda tworzenia biofilmu zależy od rodzaju Archaea i środowiska, w którym naturalnie żyje. Przybliżamy również zjawisko quorum- -sensing, jako mechanizm komunikacji Archaea w środowisku. Za zjawisko to odpowiadają cząsteczki chemiczne podobne do tych u bakterii, a mianowicie acylowany lakton homoseryny, peptyd QS, autoinduktor-2 i -3 oraz inne. Opisywane zjawiska, zarówno tworzenie biofilmu jak i quorum-sensing są istotne dla życia archeonów. Opisane zjawiska wydają się konserwatywne, ponieważ zarówno u archeonów, jak i bakterii są regulowane przez te same mechanizmy.

35 Małgorzata Pawlikowska-Warych, Beata Tokarz-Deptuła, Paulina Czupryńska, Wiesław Deptuła

Introduction

Archaea form the domain, phylogenetically distinct from the other two domains, Bacteria and Eukarya (Staley, Caetano-Anolles, 2018). The concept of this domain was proposed after research published in the 70 years of the XX century by Woese and Fox (1977). They determining the phylogenetic similarity of microorganisms, using the 16S rRNA nucleotide sequence and showed that methanogenic organisms are not bacteria, as previously believed (Forterre, Brochier, Philippe, 2002; Woese, Fox, 1977). Further investigation proved that some microorganisms from extreme ecological niches have so significant morphological, genetic, and physiological differences that they should constitute a separate taxonomic unit – domain (Forterre et al. 2002; Woese, Fox, 1977). The fact that such organisms occupied different environments, including extreme niches, which suggested their archaic origin, they have been referred to as Archaea (DeLong, 1998). Bergey’s systematic (Boom, Castelholz, 2001) differentiates Archaea into two types: Euryarchaeota and Crenarchaeota (Boom, Castelholz, 2001; Forterre et al., 2002). After a result of isolation of rRNA from Yellowstone hot springs, the systematics of the Archaea has been extended by two not classification in any taxonomy range –Korarchaeota (Effenberger,Brzezińska-Błaszczyk, Wódz, 2014; Probst, Auerbach, Moissl-Eichinger, 2013) and Nanoarchaeota (Huber, Hohn, Stetter, Rachel, 2003; Probst et al., 2013). The last one is formed by small cocci – Nanoarchaeum equitans, bound to other Archaea from the Ignicoccus genus – isolated an undersea hydrothermal vent off the coast of Iceland (Huber et al., 2003; Probst et al., 2013). Furthermore, genetic analysis of mesophilic Archaea: Cenarchaeum symbiosum, Nitrosopumilus maritimus, and Nitrososphaera gargensis, of Crenarchaeota type, revealed they should be describe alone, and now are include in not classification in range Thaumarchaeota (Spang et al., 2010). Archaea are present in various environments, including hot and cold waters, geysers, bottom sediments and soil (Boom, Castelholz, 2001; Effenbergeret al., 2014; Huyhn, Verneau, Levasseur, Drancourt, Aboudharam, 2015). They can be halo-, acido-, and basophiles (Boom, Castelholz, 2001; Effenberger et al., 2014; Huyhn et al., 2015). They acquire energy from various inorganic compounds, such as sulphur compounds, metal ions, and organic compounds: saccharides and proteins (Boom, Castelholz, 2001; Forterre et al., 2002). Some Archaea of Euryarchaeota type are capable of producing methane (Bapteste, Brochier, Boucher, 2005). In the cellular membrane of all Archaea, there is glycerol-1-phosphate, to which branched isoprenoid chains are connected using ether bonds that are stronger than ester bonds. Such organisation of cellular membrane is of importance for such microorganisms’ survival in extreme environments (Efenberger et al., 2014). In Haloquadratum walsabyi, additional external structures have been found, such as polysaccharide envelopes or halomucin capsules that help to survive in unfavourable conditions (lack of water) (Bapteste et al., 2005; Efenberger et al., 2014). Archeons, besides the extreme niches in which they occur and with which they are associated, are also a component of the plankton of the seas and oceans where they live in cooperation with environmental bacteria (DeLong, 1998). Archaea can also be a part of mammals microbiome, especially anaerobic, methanogenic genera (Methanobrevibacter, Methanobacterium, Methanosphaera), halophiles from the Halobacteriaceae family, thermophiles from Sulfolobus genus, as well as nitrification microorganisms from Nitrosphaera genus (Dridi, Didier, Drancourt, 2011; Matarazzo et al., 2012; Probst et al., 2013).

36 Biofilm and Quorum Sensing inArchaea

Biofilm inArchaea

Similarly to bacteria, Archaea can form a biofilm, multicellular communities, formed on the surfaces or interfaces of materials both organic and inorganic nature. Biofilm microorganisms participates in the circulation of many elements in the environment, and allows for degradation of harmful compounds (Caderon et al., 2013). It has been described in psychrophiles, thermophiles, halophiles, and acidophilic Archaea, as well as in organisms oxidising ammonia, and producing methane (Bapteste et al., 2005; Caderon et al., 2013; Chudy, Jabłoński, Łukaszewicz, 2011; Fernandez, Diaz, Amilis, Sanz, 2008; Mayerhofer, Macario, Conway de Macario, 1992; Ozuolmez et al., 2015; Raskin, Rittmann, Stahl, 1996). In contrast to bacteria in Archaea, biofilm formation response to stress is exceptional, because this phenomenon has only been described in Sulfolobus species during intense UV radiation (Ajon et al., 2011; Fröls, 2013) and in Haloferax volcanii exposure to lethal concentrations of biocidal compounds (Megaw, Gilmore, 2017). Similarly as in bacteria, in Archaea first steps in biofilm formation is adhere to the surface where the biofilm will be formed and then secretion of polymeric compounds outside cells (Caderon et al., 2013; Fröls, 2013). As regards extracellular polymeric substances (EPS), they have been found to include: polysaccharides, eDNA (extracellular DNA), glycosylated proteins, lipids, and enzymes specific to this group of microorganisms (Flemming, Wingender, 2010). Especially this element of biofilm being recorded in thermo-acidophilic Sulfolobus (S.) acidocaldarius, S. solfataricus, and S. tokodai (Jachlewski et al., 2015; Koch, Rudolph, Moissl, Huber, 2006). It was also evidenced that the most frequent component of polymeric compounds are the monosaccharides that glycosylate proteins (Koerdt et al., 2012; Zolghard et al., 2010), which would point to their role as nutrient storage, similarly as in the case of bacteria (Zolghard et al., 2010). As regards S. solfataricus, genes were found that are in charge of biodegradation of saccharides with β-galactosidase (LacS) and α-mannosidase (Ssα-man) activity, and it was proven that the products of such genes can condition EPS volume in biofilm, and probably control the saccharide volume in biofilm matrix (Koerdt et al., 2012). In biofilm matrix of halophiles from Halobacterium, Haloferax, and Halorobrum genera found glycosylated compounds and eDNA (Fröls, Dyall-Smith, Pfeifer, 2012) whereas in hyper-thermophile Thermococcus litoralis, biofilm matrix was formedof polysaccharide – man- nan (Rinker, Kelly, 1996). In formation of Archaea biofilm a major role belongs to extracellular organelle, such as archaella, pili – showing a different structure from the bacterial one, cannulae, ‘hami’ (Efenberger et al., 2014; Ng, Zolghadr, Driessen, Albers, Jarrell, 2008) and extracellular polymeric substances (EPS) (Flemming, Wingender, 2010). Archaella are in charge of motion, analogically to bacterial flagella. Archaellum’s filament forms a right-hand helix with the diameter of 10-14 nm (Lassak et al., 2012), although their structure reveals greater phylogenetic affinity to the bacterial type IV pili than flagella (Bardy, Jarrel, 2003).The method of secreting proteins of bacterial and archaella’s pili is similar. Archaella in Archaea are coded by the fla gene family (Bardy, Jarrel, 2003; Szabo et al., 2007). Apart from allowing motion and binding microorganisms to abiotic surfaces, archaella also play a role in the contact among such microorganisms (Lassak et al., 2012; Ng et al., 2008). Pili in Archaea are built of two sub-units that may form a helix or a ring (Pohlschröder, Esquivel, 2015; Wang, Yu, Ng, Jarrell, Egelman, 2008). There are two types of such organella, namely Ups pili and Aap pili. Ups pili occur in Sulfolobus (S.) solfataricus in the case of intense UV radiation, and participating in formation of cell batteries to accelerate DNA repair (Wang et al., 2008). Aap pili have been reported in S. acidocaldarius, which Archaea occur in environments with large volumes of nutrients, and their role is related to cell adhesion to various surfaces

37 Małgorzata Pawlikowska-Warych, Beata Tokarz-Deptuła, Paulina Czupryńska, Wiesław Deptuła and biofilm maturation (Wang et al., 2008). Cannulae are organella characteristic exclusively of Archaea from Pyrodictium genus, they participate in cell adhesion and forming biofilm (Horn, Paulmann, Kerlen, Junker, Huber, 1991; Montgomery, Charlesworth, LeBard, Visscher, Burns, 2013; Nickell, Hegerl, Baumeister, Rachel, 2003). Cannualae are built by three glycoproteins with the structure of empty tubes branching from peri-plasmatic space and they reveal resistance to high temperatures (Horn et al., 1991; Nickell et al., 2003). Cannulae have also been observed after cell division, when the daughter cells are connected by them; by multiple repetition, a colony of cells looks like connected with a network. This suggests that such organella can take part in the transmission of nutrients and the genetic material (Horn et al., 1991; Montgomery et al., 2013; Nickell et al., 2003). ‘Hami’ have been described for Altiarchaeum hamiconexum (formely SM1 euryarchaeon), that inhabit cold mashes in Bavaria that are rich in sulphur compounds (Hennenberger, Moissl, Amann, Rudolph, Huber, 2006; Moissl, Rachel, Briegel, Engelhardt, Huber, 2005). ‘Hami’ are helical tabs of about 1-3 µm with spikes spaced about every 46 nm and ended with three hooks. Such structures resemble a barbed wire have resistant to mechanical, thermal, and chemical agents, and was responsible for Archaea adhere to different surface (Hennenberger et al., 2006; Moissl et al., 2005). In Archaea inhabiting the natural environment, biofilm is popular and principally refers to Archaea from Euryarchaeota and Crenarchaeota types, formed together with bacteria (Fröls, 2013; Hennenberger et al.; 2006; Jachlewski et al., 2015; Koch et al., 2006; Koerdt, Godake, Berger, Thormann, Albers, 2010; Lapaglia, Hartzell, 1997; Rinker, Kelly, 1996; Schopf, Wanner, Rachel, Wirth, 2008; Schrenk, Kelley, Delaney, Baross, 2003; Szabo et al., 2007). In water reservoir sediments and densified water was evidenced that methanogenic Archaea, belonging to Euryarchaeota forming biofilm with bacteria that reduce sulphates from Desulfovibrionaceae and Desulfobacteriaceae. This organisms proliferated in the presence of acetates, which are a substrate for their energy (Ozuolmez et al., 2015; Raskin et al., 1996). The microorganisms occur anaerobic environment of biological wastewater treatment plants, rich in calcium and magnesium ions (Caderon et al., 2013; Chudy et al., 2011; Fernandez et al., 2008; Mayerhofer et al., 1992). Aggregates of Archaea form on solid waste particles, and can float freely on wastewater surface, or adhere to different materials (eg. polysty- rene or glass). Apart from methanogenic microorganisms of Euryarchaeota type, such biofilm is also formed by such bacteria as Bacillus sp., Aeromonas sp., Acinetobacter sp., Delftia sp., Comanomonas sp., Chryseobacterium sp., Trichococcus sp., and Nostocoida sp. (Caderon et al., 2013; Chudy et al., 2011; Fernandez et al., 2008; Mayerhofer et al., 1992). A biofilm was also reported in Ferroplasma acidarmanus, Euryarchaeota type, formed together with Leptospirillum sp. bacteria, found in acid leachate from Iron Mountain mine, Michigan (USA) (Baker-Austin, Potrykus, Wexler, Bond, Dopson, 2010). As regards biofilm formed by Ignisphaera aggregans and Pyrobaculum sp. of Crenarcheota type, isolated from the slide of a pool located near hot springs in New Zealand, it was found that the biofilm can be formed as aggregates, particularly with polysaccharides present in subgrade, as they form the source of carbon (Niederbeger, Götze, McDonald, Ronimus, Morgan, 2006). In case of Nitrosopumilus maritimus was reported that bioflim formed with bacteriaof Nitrosomonas and Nitrospira genera on a polyvinyl trickling filter in marine aquaculture system (Foesel et al., 2008). If was revealed that, in the process of adhesion to glass plates, Methanopyrus (M.) kandleri and Pyrococcus (P.) furiosus form multilayer biofilms using pili, and probably also archaeallum (Rinker, Kelly, 1996). Thermococcus (T.) litoralis and P. furiosus, however, together with other hyperthermophiles, form biofilm on glass and polycar- bonate plates, although the time of biofilm formation depends on the presenceof maltose or yeast extract (Rinker, Kelly, 1996). In geothermal conditions, namely in hot springs and hydrothermal

38 Biofilm and Quorum Sensing inArchaea vents in seas, biofilm has also been reported in hyper-thermophile M. kandleri and T. litoralis from Euryarchaeota, which form biofilm with P. furiosus (Rinker, Kelly, 1996; Schopf et al., 2008). Biofilm mixed with bacteria is also formed by Euryarchaeota from Methanosarcinales, Thermococcales, Archaeoglobales orders, and Archaea from Thermoproteales order, Cranarchaeota type, isolated from hydrothermal vents with high content of sulphur compounds (Schrenk et al., 2003). Organisms using filaments can form aggregates on mineral deposits largely formed of sulphur compounds (Schrenk et al., 2003). It was observed that hyperthermophiles from Archaeoglobus fulgidus, belonging to Euryarchaeota type and inhabiting seals, is the only representatives of Archaea that forms biofilm composedof polysaccharides, proteins, and metal ions. This biofilm is formed after activationof stress factors, such as UV radiation, temperature change, pH decrease, or increased concentration of metals and chemotherapeutic agents (Fröls, 2013; Lapaglia, Hartzell, 1997; Ng et al., 2008). The biofilm formation has also been observed in such Archaea as: Halobacterium salinarum, Haloferax volcanii, Halorubrum lacusprofundi, and the Halohasta litchfieldiae (strain DL24) (Fröls et al., 2012). Biofilm of Halohasta litchfieldiae is formed on abiotic surfaces by archaeallum and EPS, principally eDNA and glycosylated organic compounds, and has the structure of a single layer or aggregate structure (Haloferax volcanii, or Halorubrum lacusprofundi) (Fröls, 2013). Also psychrophilic Archaea of Euryarchaeaota type, Altiarchaeum hamiconexum, in aquatic environment with high concentration of sulphuric compounds forming biofilm together with bacteriaThiothrix sp. on polyethylene strands using ‘hami’ (Bird, Baker, Probst, Podar, Lloyd, 2016; Hennenberger et al., 2006). The biofilm is characterised with a structure resembling a pearl necklace, where the external layer is formed by bacteria, while the internal one by Archaea bound to the strand (Hennenberger et al., 2006). Biofilm has also been reported in Archaea of Crenarchaeota type, including S. solfataricus, S. acidocaldarius, and S. tokodai species that inhabit geothermal areas with volcanic activity (Jachlewski et al., 2015; Koerdt et al., 2012, Koerdt et al., 2011; Szabo et al., 2007). The biofilm is formed using archaellum and pili, and with saccharide secretion. Moreover, in laboratory conditions, biofilm formation on pilus-dependent way, upon UV exposure was found S. solfataricus and S. acidocaldarius (Jachlewski et al., 2015; Koerdt et al., 2012; Pohlschröder, Esquivel, 2015; Szabo et al., 2007). Other thermo-acidophiles – Metallosphaera sedula from Crenarchaeota type, with the capacity of iron compound oxidation, forming biofilm as aggregates using archaellum, on surfaces containing iron sulphide. The biofilm formation process probably involves EPS, glycosylated using ga- lactose or ramnose (Auernik, Maezato, Blum, Kelly, 2008).

Quorum sensing in Archaea

Quorum sensing (QS) is a cell to cell signaling mechanism among bacterial cells which coordinate their activities. In this mechanism many various species-specific QS signaling molecules (QSSMs) are used. Acylated homoserines lactones (AHLs), QS peptides (QSPs), autoinducer-2 (AI-2), diketopiperazines (DKPs), autoinducer-3 (AI-3) and another are included for these molecules (Rajput, Gupta, Kumar, 2015; Rajput, Kaur, Kumar, 2016). The phenomenon, both in bacteria and Archaea is conditioned with genes that allow their adaptation and survival in the unfavourable environment, such as temperature shock, lack of nutrients, drastic changes to salinity and pH, and the presence of chemotherapeutical agents (Bassler, 2002; Megaw, Gilmore, 2017; Montgomery et al., 2013; Paggi, Martone, Fuqua, De Castro, 2003). In the case of the Bacteria domain, the QS mechanism can be divided into three types depending on autoinducer-1 (AI-1), peptide and autoinducer-2 (AI-2), wherein systems based on AI-1 and AI-2 are common

39 Małgorzata Pawlikowska-Warych, Beata Tokarz-Deptuła, Paulina Czupryńska, Wiesław Deptuła in Gram-negative bacteria, and peptide based system occurs in Gram-positive bacteria (Barriuso, Martinez, 2018; Kaur, Capalash, Sharma, 2018; Montgomery et al., 2013). The systems AI-1 and AI-2 use AHLs (acylated homoserine lactones) as inducers, where system AI-1 use LuxR and LuxI proteins, and system AI-2 use proteins like LuxS, LuxP, LsrB, RosB, Pfs and SAH hydrolase (Abisado, Benomar, Klaus, Dandekar, Chandler, 2018; Montgomery et al., 2013). In AI-1 system, LuxI produce N-3-oxo-hexanoyl-homoserine lactone, which specifically binds to LuxR, which is a factor that activates luxCDABEG operon, which contains for examples the genes enables bioluminescence (Abisado et al., 2018) and the genes that condition bacterial survival in the new conditions (Bassler, 2002; Myszka, Czaczak, 2010). Most of bacterial AHLs are produced from S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway (Abisado et al., 2018). In Gram-positive bacteria, the inductor function is held by short oligopeptides formed as a result of processing longer protein chains secreted to the environment via active transport. When the concentration of such compounds (inductors) reaches a critical level, this is recognised by a specific receptor: histidine kinase, and a number of reactions occur aimed at formation of a regulatory protein that would affect the expression of target genes (Jaworski, Serwecińska, Stączek, 2005). In the case of Archaea, a phenomenon analogical to the bacterial QS system was reported for haloalkaliphilic archaeon Natronococcus occultus (Euryarchaeota type), where it was observed that together with the increase in culture density and exhaustion of nutrients in the medium, the volume of proteases secreted outside the cells increases. Research focused at identifying this phenomenon did not manage to point to AHL-like self-inductors due to their sensitivity to alkaline pH and the presence of short chains of such particles (Montgomery et al., 2013; Paggi et al., 2003). Analogical research referred to Natrialba magadii, of Euryarchaeota type, which occupies the same ecological niches as Natronococcus occultus and revealed that AHL-like particles can act as inductors, but only when their chain is long enough for the inductor to remain stable in alkaline environment (Paggi et al. 2003; Paggi, Madrid, D’Alessamdro, Cerletti, De Castro, 2010). Communication among Archaea cells, using an AHL-like system, was at the first time evidenced forMethanosaeta harundinacea (Euryarchaeota type) where three compounds were detected: N-carboxyl-decanoyl-homoserin lactone, N-carboxyl-dodecanoyl-homoserine lactone and N-carboxyl-tetradecanoyl-homoserine lactone (Zhang et al., 2012). Those compounds are formed as a result of activation of filI-filR genes being orthologs of the bacterial luxI-luxR system (Zhang et al., 2012). Research focused on the QS system in Methanosaeta harundinacea has proved that the system is responsible for filament production and changes to carbon metabolism (Li et al., 2015; Montgomery et al., 2013). It was also evidenced that cyclo-(L-prolyl-L-valine), belonging to the diketopiperazines (DKPs) can act as signal particles in QS phenomenon in Haloterrigena hispanica (Euryarchaeota type), unrelated to the typical bacterial communication system (Tommonaro, Abbamondi, Iodice, Tait, De Rosa, 2012). In the culture of those halophiles, five variants of diketopiperasines (DKPs) particles were extracted, which can participate in the adaptation of such Archaea in extremely salinated environment and, together with bacterial AHL, in the communication of mixed cultures in the biofilm (Tommonaro et al., 2012). Also in halophilic strain SK-6, belonging to Haloterrigena genus, compounds of diketopiperazines was found (Abed, Gotthard, Champion, Chabriere, Elias, 2013). The lactonase VmoLac, phosphotriesterase-like lactonase (PLL) was reported in hyperthermophile Vulcanisaeta moutnovskia (Crenarcheota type) (Hiblot et al., 2013). This lactonase hydrolyze various lactones, including AHLs, what suggest that could be involved in QS in this archaeon (Hiblot, Bzdrenga, Champion, Chabriere, Elias, 2015). Also in Pyroccocus furiosus, hypertermophilic archaeon, was observed that temperature mediates autoinducer-2 (AI-2)

40 Biofilm and Quorum Sensing inArchaea formation, despite lacking the enzymes Pfs and LuxS (Nichols, Johnson, Chou, Kelly, 2009). It evidence, that Archaea use QS mechanism typical for bacteria, but using different molecules to activate the signalling pathways (Montgomery et al., 2013; Nichols et al., 2009). In the last research was found that only SAH hydrolase (S-adenosyl-L-homocysteine hydrolase) and Pfs (5’methylthioadenosine/S-adenosylhomocysteine nucleosidae), a part of AI-2 system are presence in termophiles Archaea (Kaur et al., 2018).

Conclusion

Archaea, are microorganisms that inhabit very different environments, from ecological niches as. hypersaline, hot and cold waters, marine sediments, soil, and even mammalian microbiome. They are known from over 40 years but still new facts are discovered, e.g. about biofilm formation and cell-to-cell signaling mechanism. It is know that Archaea’s biofilm is formed by polymeric particles secreted by such microorganisms to the biological membrane matrix, which sustains interactions among organisms. Archaea form biofilm together with bacteria, thus they participate in the transformation of various compounds and play a major role in the environment, such as biological wastewater treatment processes. Communication mechanism for organisms in the domain is similar to the bacterial quorum sensing, but occurring using a little bit different signal particles. This facts about the Archaea group reveal new data in the area of their physiology.

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Cite as: Pawlikowska-Warych, M., Tokarz-Deptuła, B., Czupryńska, P., Deptuła, W. (2019). Biofilm and Quorum Sensing in Archaea. Acta Biologica, 26, 35–44. DOI: 10.18276/ab.2019.26-04. #1#

44 #0#

Acta Biologica 26/2019 | www.wnus.edu.pl/ab | DOI: 10.18276/ab.2019.26-05 | strony 45–55

Somatic and F-specific bacteriophages in waters of the small, municipal Rusałka Lake in Szczecin

Małgorzata Pawlikowska-Warych,1 Paulina Czupryńska,2 Beata Tokarz-Deptuła,3 Wiesław Deptuła4

1 Department of Microbiology, Faculty of Biology, University of Szczecin, Felczaka 3c, 71-412 Szczecin, Poland 2 Department of Microbiology, Faculty of Biology, University of Szczecin, Felczaka 3c, 71-412 Szczecin, Poland 3 Department of Immunology, Faculty of Biology, University of Szczecin, Felczaka 3c, 71-412 Szczecin, Poland 4 Nicolaus Copernicus University in Toruń, Faculty of Biological and Veterinary Sciences, Institute of Veterinary Medicine, Gagarina 7, 87-100 Toruń, Poland

Corresponding author, e-mail: [email protected]

Keywords Bacteriophages, aqueous environment, environment protect Abstract Investigations of bacteriophages showed that in the environment are mostly in water, but in small municipal lakes sporadically been studied. Previous studies of bacteriophages in the aqueous environment related to the determination of the quantity and diversity and use them as indicators of water pollution. In the last study probably of the most important are the F-specific and somatic bacteriophages specific toE. coli. In our study examined them in a small municipal lake in Szczecin (Poland) in context of occurrence of F-specific and somatic bacteriophages specific to E. coli with used of SAL method. We proved that count of bacteriophages in this water tank is lower in context of different waters, but the relation between the amounts of this viruses and environments condition (air and water temperature) were similar to results observed in waters of seas, rivers and lakes.

Somatyczne i F-specyficzne bakteriofagi małego miejskiego jeziora Rusałka w Szczecinie Słowa kluczowe bakteriofagi, środowisko wodne, ochrona środowiska Streszczenie Badania bakteriofagów wykazały, że w środowisku naturalnym znajdują się one głównie w wodzie, ale w małych jeziorach miejskich badano je sporadycznie. Poprzednie badania bakteriofagów w środowisku wodnym dotyczyły określania ilości i różnorodności oraz wyko- rzystywania ich jako wskaźników zanieczyszczenia wody. Wcześniejsze badania wykazały, że prawdopodobnie najważniejszymi są F-specyficzne i somatyczne bakteriofagi E. coli. W obecnym badaniu oznaczaliśmy je w małym jeziorze miejskim w Szczecinie (Polska), za pomocą metody SAL. Udowodniliśmy, że liczba bakteriofagów w tym zbiorniku jest mniejsza w porównaniu do innych wód, ale związek między liczbą tych wirusów a warunkami środo- wiska (temperatura powietrza i wody) był podobny do wyników obserwowanych w wodach mórz, rzek i jezior.

45 Małgorzata Pawlikowska-Warych, Paulina Czupryńska, Beata Tokarz-Deptuła, Wiesław Deptuła

Introduction

Nonillion (10^30) viruses are estimated to exist in the world (Paez-Espino et al., 2016), most of which are bacteriophages occurring in aqueous environment (Breitbart, Rohwer, 2005; Paez- Espino et al., 2016). Research into bacteriophages in aqueous environments was conducted on flowing (Brazina, Baldini, 2008; Burbano-Rosero et al., 2011; Cole, Long, Sobsey, 2003; Dore, Henshillwood, Lees, 2000; Duran et al., 2002; Fauvel, Gantzer, Cauchie, Ogorzaly, 2016; Havelaar, van Olphen, Drost, 1993; Hernroth, Conden-Hansson, Rehnstam-Holm, Girones, Allard, 2002; Hot, Legeay, Jacques, Gantzer, Caudrelier, Guyard, 2003; Jiang, Paul, 1994; Jiang, Noble, Chu, 2001; Jiang, Chu, He, 2007; Long, Sobsey, 2004; Moce-Livina, Lucena, Jofre, 2005; Ogorzaly, Tissier, Bertrand, Maul, Ganzer, 2009; Shirasaki, Matsushita, Matsui, Urasaki, Ohno, 2009; Simkova, Cervenka, 1981; Sinton, Finlay R. K., Lynch, 1999; Skraber, Gassilloud, Gantzer, 2004; Stewart et al., 2006; Stewart-Pullaro et al., 2006; Zupok, Sokołowska, Śliwa-Dominiak, Tokarz-Deptuła, 2010) and standing waters, but mainly on large lakes (Demuth, Neve, Witzel, 1993; Drucker, Dutova, 2006; Dryden, Ramaswami, Yuan, Giammar, Angenet, 2006; Havelaar et al., 1993; Hennes, Simon, 1995; Jiang, Paul, 1994; Pusch et al., 2005; Stewart, Vinje, Oudejans, Scott, Sobsey, 2006; Stewart-Pullaro et al., 2006; Śliwa-Dominiak, Tokarz-Deptuła, Deptuła, 2010, 2014; Zupok et al., 2010), the water of seas and oceans (Dore et al., 2000; Hernroth et al., 2002; Jiang, Paul, 1994; Jiang et al., 2007; Moce-Livina et al., 2005; Rava, Sarreal, 2016; Sinton et al., 1999; Stewart et al., 2006; Suttle, 2007) and drinking water (Pelleieux, Mathieu, Block, Gantzer, Bertramd, 2016; Vergara, Goh, Rezaeinejad, Sobsey, Gin, 2015). Despite an essential role municipal lakes play relative to their location in a city and their recreational character, there exists little body of literature on phages found in the water of small lakes, including municipal lakes. Those papers focused on somatic bacteriophages (Brazina, Baldini, 2008; Burbano-Rosero et al., 2011; Demuth et al., 1993; Dore et al., 2000; Hennes, Simon, 1995; Hot et al., 2003; Jiang, Paul, 1994; Moce-Livina et al., 2005; Pusch et al., 2005; Sinton et al., 1999; Skraber et al., 2004; Śliwa-Dominiak et al., 2014; Zupok et al., 2010), F-RNA (Cole et al., 2003; Demuth et al., 1993; Dore et al., 2000; Dryden et al., 2006; Duran et al., 2002; Fauvel et al., 2016; Havelaar et al., 1993; Hennes, Simon, 1995; Hernroth et al., 2002; Jiang, Paul, 1994; Jiang et al., 2001; Jiang et al., 2007; Long, Sobsey, 2004; Moce-Livina et al., 2005; Ogorzaly, Tissier, Bertrand, Maul, Ganzer,. 2009; Olson, Axler, Hicks, Henneck, McCarthy, 2005; Pusch et al., 2005; Shirasaki et al., 2009; Simkova, Cervenka, 1981; Sinton et al., 1999; Stewart et. al., 2006; Stewart-Pullaro et al., 2006; Śliwa-Dominiak et al., 2010, 2014; Vergara et al., 2015; Zupok et al., 2010) and F-DNA phages (Cole et al., 2003; Hennes, Simon, 1995; Jiang, Paul, 1994; Jiang et al., 2001; Long, Sobsey, 2004; Pusch et al., 2005; Zupok et al., 2010) and on Bacterioides fragilis bacteriophages (Moce- Livina et al., 2005; Ogorzaly et al., 2009; Skraber et al., 2004). Bacteriophages were identified in observational studies and when aqueous environments were evaluated for water pollution, phages were detected together with E. coli bacteria (Brazina, Baldini, 2008; Burbano-Rosero et al., 2011; Dore et al., 2000; Fauvel et al., 2016; Hernroth et al., 2002; Jiang, Paul, 1994; Jiang et al., 2001; Moce-Livina et al., 2005; Ogorzaly et al., 2009; Olson et al., 2005; Pusch et al., 2005; Sinton et al., 1999; Skraber et al., 2004; Śliwa-Dominiak et al., 2010, 2014; Vergara et al., 2015) and Enterococcus faecalis (Havelaar et al., 1993; Jiang, Paul, 1994; Jiang et al., 2001; Moce-Livina et.al., 2005; Ogorzaly et al., 2009; Pusch et al., 2005; Sinton et al., 1999; Skraber et al., 2004). In the latter, F-specific genogroupsof RNA bacteriophages were characterized (Cole et al., 2003; Demuth et al., 1993; Dore et al., 2000; Dryden et al., 2006; Duran et al., 2002; Fauvel et al., 2016; Havelaar et al., 1993; Hennes, Simon, 1995; Hernroth et al., 2002; Hot et al., 2003; Jiang,

46 Somatic and F-specific bacteriophages in waters of the small, municipal Rusałka Lake in Szczecin

Paul, 1994; Jiang et al., 2001; Jiang et al., 2007; Long, Sobsey, 2004; Moce-Livina et al., 2005; Ogorzaly et al., 2009; Pusch et al., 2005; Shirasaki et al., 2009; Simkova, Cervenka, 1981; Sinton et al., 1999; Stewart et al., 2006; Stewart-Pullaro et al., 2006; Śliwa-Dominiak et al., 2010, 2014; Zupok et al., 2010), as genogroups I and IV are common in zoonotic while genogroups II and III in human contamination (Cole et al., 2003; Fauvel et al., 2016; Long, Sobsey, 2004; Moce- Livina et al., 2005; Stewart et al., 2006; Stewart-Pullaro et al., 2006; Śliwa-Dominiak et al., 2014; Vergara et al., 2015). Observational studies were carried out on the effectof the number of somatic bacteriophages (Jiang, Paul, 1994; Moce-Livina et al., 2005) and F-specific RNA bacteriophages on the number of people-infecting viruses (Hernroth et al., 2002; Jiang, Paul, 1994; Jiang et al., 2001; Moce-Livina et al., 2005; Ogorzaly et al., 2009; Vergara et al., 2015). Research was done on the effectof somatic bacteriophages (Hot et al., 2003; Pusch et al., 2005; Simkova, Cervenka, 1981; Skraber et al., 2004) and F-specific RNA bacteriophages (Havelaaret al., 1993; Jiang et al., 2001; Ogorzaly et al., 2009; Pusch et al., 2005; Simkova, Cervenka, 1981; Vergara et al., 2015) on the number of viruses infecting animals. Bacteriophages in aqueous environments (Hennes, Simon, 1995; Jiang et al., 2007; Śliwa-Dominiak, Tokarz-Deptuła, Deptuła, 2015) were determined as part of the microbial loop. By killing bacteria, they promote the release of compounds of carbon, nitrogen and phosphorus into the environment, all of which are important building blocks of life, and particularly in case of plankton. Some studies focused on the impact of temperature in the environment (Cole et al., 2003; Dore et al., 2000; Fauvel et al., 2016; Hernroth et al., 2002; Jiang, Paul, 1994; Long, Sobsey, 2004; Olson et al., 2005; Ravva, Sarreal, 2016; Simkova, Cervenka, 1981; Sinton et al., 1999; Skraber et al., 2004; Śliwa-Dominiak et al., 2010, 2014), including exposure to sunlight (Cole et al., 2003; Dore et al., 2000; Fauvel et al., 2016; Hernroth et al., 2002; Sinton et al., 1999; Śliwa-Dominiak et al., 2015) on the water levels of somatic bacteriophages (Duran et al., 2002; Hernroth et al., 2002; Jiang, Paul, 1994; Simkova, Cervenka, 1981; Sinton et al., 1999; Skraber et al., 2004) and F-specific RNA bacteriophages (Coleet al., 2003; Dore et al., 2000; Fauvel et al., 2016; Hernroth et al., 2002; Jiang, Paul, 1994; Long, Sobsey, 2004; Ravva, Sarreal, 2016; Sinton et al., 1999; Śliwa-Dominiak et al., 2010, 2014; Vergara et al., 2015). They regulate E. coli levels which determine water clarity. As there is no data on bacterial viruses in the water of small, municipal lakes other that collected by our team on FRNA phages (Śliwa-Dominiak et al., 2010, 2014; Zupok et al., 2010), we identified somatic and F-specific RNA and DNA bacteriophages in the waterof a municipal Lake Rusałka in Szczecin, within the framework of four seasons (spring, summer, autumn, winter), understood to be an element conditioning the effect of water and air temperature on the amount of examined bacteriophages.

Experimental procedures

Rusałka Lake, with its area of 3.7 hectares, is located in the centre of Szczecin. It is elongated in shape, 670 m long and approximately 40 m wide (at its widest point). It is a shallow reservoir of the max depth of 2 m and it is substantially silted. It is a flow-through reservoir as it is fed by the water of Osówka stream and surplus water can escape through an underwater pipeline running through Niecka Niebuszewska and Stocznia Szczecińska (Szczecin Shipyard) into the West Oder (Hłynczak, Deptuła, Możdżer, Poleszczuk, Słupińska, 1995). Water samples were taken once at two points: at the inlet of the stream (point A) and at the opposite direction, at its outlet (point B). The distance between points A and B was approximately 670 m. Samples were taken in four seasons; spring, summer, autumn and winter. Water sampling, water and air temperature

47 Małgorzata Pawlikowska-Warych, Paulina Czupryńska, Beata Tokarz-Deptuła, Wiesław Deptuła measurement procedures complied with PN-EN ISO 19458 standards (19 December 2007, Polish Committee for Standardisation). The amount of somatic and F-specific RNA and DNA bacteriophages identified in water samples taken at points A and B in spring, summer, autumn and winter was determined with Single Agar Layer SAL (Annon: Method 1602: Male- specific (F+) and Somatic Coliphage in Water by Single Agar Layer (SAL) Procedure. 2001, EPA, Washington D.C.). In bacteriophages plaque assay, phages were determined as the number of plaque forming units (PFU) in ml of examined water. Different strains of Escherichia (E.) coli were used to detect and determine phages concentration. Nalidixic acid-resistant E. coli CN13 was used for somatic bacteriophages while ampicillin and streptomycin-resistant E. coli Famp strain was used for F-specific RNA and DNA bacteriophages. To obtain clean cultures, commercial strains of E. coli CN13 and E. coli Famp were in line with the requirements grown on Tryptic Soy Agar – TSA solid medium, inoculated with an antibiotic. Each colony was then transferred to TSB liquid medium and each was grown in the presence of antibiotics it was resistant to. To identify somatic bacteriophages and F-specific RNA and DNA phages, medium consisted of 100 ml of water taken in the sampling points, 100 ml of double concentrated TSA (Triptic Soya Agar), 0.5 ml of sterile solution of MgCl2x6H2O with a concentration of 0.0814 g/ml, 2 ml of antibiotic (Nalidixic acid for E. coli CN13 and a mixture of ampicillin and streptomycin for E. coli Famp) and 10 ml of 24-hour broth culture (E. coli CN13 or E. coli Famp). All constituent elements were mixed and transferred to Petri dishes which were incubated for 24 h at 37°C and PFUs were counted. Results from bacteriophage plaque assay of somatic bacteriophages and F-specific RNA and DNA phages in the samplesof water taken in points A and B, considering their dependence on temperature of water and air and resulting from seasonal changes were used for statistical analysis (Kończak, Trzpiot 2002). Sperman’s rank correlation coefficient was calculated. It determines dependence (correlation) between examined variables and it has a value in the interval (–1, 1). The method (Ostasiewicz, Rusnak, Siedlecka, 2003), which is used for correlation power determination, assumes that if its value is in the range of 0–0.2, it means weak correlation, 0.2–0.4 low correlation, 0.4–0.7 moderate correlation, 0.7–0.9 significant correlation and over 0.9 very strong correlation.

Results

Analysis of somatic bacteriophages and F-specific RNA and DNA phages in the samples of water taken in points A in spring, summer, autumn and winter showed the levels of somatic bacteriophages in spring, in water temperature of 7°C and air temperature of 9°C was 0,3 PFU ml-1 of water and the level of F-specific RNA and DNA phages was 0,87 PFU ml-1 of water. In summer, at 13°C in water and 20°C in the air, results were 6,06 PFU ml-1 of water for somatic bacteriophages and 23,87 for F-specific RNA and DNA phages. In autumn, at 12°C in water and 8.5°C in the air, results were 21,08 for somatic bacteriophages and 0,65 PFU ml-1 of water for F-specific RNA and DNA phages.In winter, at 3°C in water and –3°C in the air, results were 20,85 PFU ml-1 of water for somatic bacteriophages and 1,17 PFU ml-1 of water for F-specific RNA and DNA phages (Table 1).

48 Somatic and F-specific bacteriophages in waters of the small, municipal Rusałka Lake in Szczecin

Table 1. The count of somatic and F-specific DNA and RNA bacteriophages in water samples from Rusałka lake and temperatures of water and air in point A and B

Count of somatic Count of F-specific Temperature Temperature bacteriophages bacteriophages of water of air Season of water samples taken (PFU ml-1) (PFU ml-1) (°C) (°C) A B A B A B A B Spring 0.300 0.130 0.870 0.000 7.0 7.6 9.0 9.0 Summer 6.060 0.170 23.870 0.000 13.0 18.0 20.0 17.0 Autumn 21.080 0.720 0.650 0.050 12.0 14.8 8.5 9.0 Winter 20.850 3.870 1.170 0.060 3.0 0.0 –3.0 –4.0 The average count of bacterio- 12.073 1.223 6.640 0.0275 8.8 10.1 8.6 7.8 phages in 4 seasons of year Explenation: PFU – plaque forming unit; A, B – water points.

In summary, the maximum and minimum levels of somatic bacteriophages were in autumn and winter and in spring, respectively at point A in four seasons. The maximum and minimum levels of F-specific RNA and DNA phages were in summer and autumn, respectively. However, the average count of somatic bacteriophages, in four seasons, at point A relative to F-specific RNA and DNA phages was higher twice, 12,073 PFU ml-1 of water for somatic bacteriophages. The average count of F-specific RNA and DNA phages was 6,64, for average water and air temperature for four seasons of 8.8°C and 8.6°C, respectively. A similar phages analysis at point B showed 0,13 PFU ml-1 for somatic bacteriophages, at water temperature of 7.6°C and air temperature 9.0°C. No F-specific RNA and DNA phages were found. In summer, at water temperature of 18°C and air temperature of 17°C, the number of somatic bacteriophages was 0,17 PFU ml-1. Just like in spring, no F-specific RNA and DNA phages were found. In autumn, at water temperature of 14.8°C and air temperature of 9°C, the number of somatic bacteriophages and F-specific RNA and DNA phages was 0,65 PFU ml-1 of water and 0,05 PFU ml-1, respectively. In winter, at water temperature of 0°C and air temperature of –4°C, the number of somatic bacteriophages and F-specific RNA and DNA phages was 3,87 PFU ml-1 of water and 0,06 PFU ml-1, respectively. To sum up results of samples taken at point B of the investigated lake, in four seasons, the maximum and minimum levels of somatic bacteriophages were in winter and in spring, respectively. The maximum levels of F-specific RNA and DNA phages were in autumn and winter, and in spring and summer there was no bacteriophages. However, the average count of somatic bacteriophages, in four seasons, at point B relative to F-specific RNA and DNA phages was 60 times higher, 1.223 PFU ml-1 for somatic bacteriophages. The average count of F-specific RNA and DNA phages was 0.0275, for average water and air temperature for four seasons of 10.1°C and 7.8°C, respectively. To evaluate the number of phages at points A and B, in four seasons, the maximum levels of somatic bacteriophages and F-specific RNA and DNA phages were found at point A: in autumn and winter for the former and in summer for the latter. The minimum levels of somatic bacteriophages and F-specific RNA and DNA phages were found at point A: in spring for the former and in spring and summer for the latter. To evaluate the averaged number of the phages, for four seasons, at points A and B where samples were taken, the number of somatic bacteriophages at point A was 10 times higher compared to point B and that of F-specific RNA and DNA phages

49 Małgorzata Pawlikowska-Warych, Paulina Czupryńska, Beata Tokarz-Deptuła, Wiesław Deptuła was over 200 times higher at point A compared to point B. Water average temperature, for four seasons, at point A compared to point B was 1.3°C lower and that of air was 0.8°C higher. Correlation between somatic bacteriophages number and water and air temperature at point A was negative (Table 2).

Table 2: Sperman’s rank correlation coefficient (a) and correlation power (b) between count of somatic or F-specific (DNA and RNA) bacteriophages and temperatures of water and air in point A and B.

Point A Point B count of somatic count of F-specific count of somatic count of F-specific bacteriophages bacteriophages bacteriophages bacteriophages (PFU ml-1) (PFU ml-1) (PFU ml-) (PFU ml-1) a) 0.59524; a) –0.80786; a) –0.48188; Temperature a) –0.17809; b) moderate b) significant b) moderate of water (°C) b) weak correlation correlation correlation correlation a) –0.59279; a) 0.79745; a) –0.91992; a) –0.76944; Temperature b) moderate b) significant b) very strong b) significant of air (°C) correlation correlation correlation correlation Explenation: PFU – plaque forming unit; A, B – water point.

Its correlation power being an absolute value of correlation coefficient of 0.17809 suggests weak correlation. However, although correlation between bacteriophage number and air temperature at point A was negative, its correlation coefficientof –0.59279 suggest moderate correlation. Correlation between F-specific RNA and DNA phage number and water temperature at point A was positive, with correlation coefficientof 0.59524 which suggest moderate correlation. Correlation between F-specific RNA and DNA phage number and air temperature at point A was positive, with correlation coefficient of 0.79745 which suggests significant correlation. A similar evaluation of correlation at point B between somatic becteriophage number and water temperature was negative, with a power of –0.80786 which suggests significant correlation. Correlation between the number of somatic bacteriophages and air temperature at the same point was negative, with a power of –0.91992 which suggests very strong correlation. Correlation between F-specific RNA and DNA phage number and water temperature was negative, with correlation coefficient of –0.48188 which suggests moderate correlation. Correlation between F-specific RNA and DNA phage number and air temperature was negative, with correlation coefficientof –0.76944 which suggests significant correlation. To sum up results of correlation between phage number and water and air temperature at points A and B, weak correlation was found only between somatic bacteriophage number and water temperature at point A. Moderate correlation was found between F-specific RNA and DNA phage number and water temperature and between somatic bacteriophage number and air temperature at point A and between F-specific RNA and DNA phage number and water temperature at point B. Significant correlation was found between F-specific RNA and DNA phage number and air temperature at point A and between somatic bacteriophage number and water temperature and between F-specific RNA and DNA phage number and air temperature at point B. Strong correlation was found between somatic bacteriophage number and air temperature at point B.

50 Somatic and F-specific bacteriophages in waters of the small, municipal Rusałka Lake in Szczecin

Discussion of results

Interpretation of data about somatic bacteriophage number and F-specific RNA and DNA phage number in the water of a small lake in Szczecin is difficult as there is no relative research conducted on similar water reservoirs other than our own studies on the number of F-RNA phages in water samples from two small lakes in Szczecin – Rusałka (Śliwa-Dominiak et al., 2010, 2014) and Syrenie Stawy (Zupok et al., 2010). However, the number of investigated F-RNA bacteriophages of 23,00 was substantially lower than that found in large water reservoirs and very large lakes of Constans (Germany), between 5,000 and 25,000 bacteriophage particles (Henne, Simon, 1995), Pluβsee (Germany), over 40 phages (Demuth et al., 1993), and Baikal (Russia), becteriophage number of 108 per ml of water (Drucker, Dutova, 2006). Based on our own data, the maximum number of somatic bacteriophages were at point A, in autumn and winter and the minimum number at point B, in spring. This is confirmed in phage levels relative to their averaged number in four seasons as phage number at point A was 10 times higher than that at point B. This is probably because at point A, the stream inlet, there are more bacteria, meaning food for phages. Our results showing higer levels of somatic bacteriophages in autumn and winter relative to spring and summer are consistent with those of Pluβsee (Germany) (Demuth et al., 1993) and Baikal (Russia) (Drucker, Dutova, 2006) where decreasing environment temperature resulted in higher phage levels. This seems to be related to the fact that in warmer months, relative to colder months, water is more exposed to UV radiation and its more intensive impact and finally to destructionof bacterial viruses (Demuth et al., 1993). The maximum number of F-specific RNA and DNA phages at point A was in summer and the minimum number at point B was in summer and spring which is consistent, similarly to somatic bacteriophages, with their number averaged for four seasons as their number was 200 times higher at point A relative to point B. That data, similarly to somatic bacteriophage data, can be related to higher levels of bacteria in water, at point A, the stream inlet to the lake. To sum up, data on F-specific RNA and DNA phage number relative to season are not fully consistent with earlier data from a study on F-RNA phages (Śliwa-Dominiak et al., 2010, 2014; Zupok et al., 2010) conducted in in the water of a similar watershed. The earlier data demonstrated higher phage levels in winter months compared to summer and our data showed their maximum levels in summer and minimum levels in autumn. This might be due to the fact that while in the recent study F-RNA and DNA phages were examines, only F-RNA phages were analysed in the earlier study. Higher levels of somatic and F-specific RNA and DNA phage numbers at point A, compared to point B, are probably due to higher numbers of microorganisms, including E. coli, in the stream at point A and due to lower temperature of water at point A, compared to point B. The latter correlation was confirmed in many other studies (Cole et al., 2003; Dore et al., 2000; Fauvel et al., 2016; Hernroth et al., 2002; Jiang, Paul, 1994; Long, Sobsey, 2004; Ravva, Sarreal, 2016; Simkova, Cervenka, 1981; Sinton et al., 1999; Skraber et al., 2004; Śliwa-Dominiak et al., 2010, 2014) in which also water temperature was demonstrated to be conducive to phage proliferation, in line with a rule of virusology that says that viruses are not very susceptible to low temperatures. However, the fact that the number of somatic phages was twice as high as that of F-specific phages confirms data from a study on the Mediterrianian Sea close to Barcelona (Moce-Livina et al., 2005), Werbeline Lake in central Germany (Pusch et al., 2005) and drinking water watershed in Singapore (Vergara et al., 2015) where also more somatic than F-specific phages were found. It is thought to be linked to the fact that somatic phages are more resistant to external factors, including exposure to sunlight, environment temperature and presence of organic matter

51 Małgorzata Pawlikowska-Warych, Paulina Czupryńska, Beata Tokarz-Deptuła, Wiesław Deptuła

(Hernroth et al., 2002; Jiang, Paul, 1994; Sinton et al., 1999). This was also confirmed in a study on the effectof temperature and salinity on bacteriophages from the estuary of a river in California (Havelaar et al., 1993), an observational study on the effect of organic matter in wastewater in Sydney (Australia) (Olson et al., 2005), a study on the effectof water temperature and chlorophyll concentration in Constance Lake (Germany) (Hennes, Simon, 1995) which demonstrated that F-specific phages were more susceptible to these factors than somatic phages. Those earlier findings regarding higher resistance of somatic phages, compared to F-specific RNA and DNA phages, to environmental conditions confirm our own data on correlation between somatic and F-specific RNA and DNA phage numbers and temperature of water and air. Stronger correlation was demonstrated between environmental conditions (air and water) and F-specific RNA and DNA phage numbers than that between environmental conditions and somatic phages. This observation was confirmed in other research studies (Coleet al., 2003; Dore et al., 2000; Fauvel et al., 2016; Hernroth et al., 2002; Jiang, Paul, 1994; Long, Sobsey, 2004; Olson et al., 2005; Simkova, Cervenka, 1981; Sinton et al., 1999; Skraber et al., 2004; Stewart et al., 2006; Śliwa-Dominiak et al., 2010, 2014) which showed that water temperature affects phage levels to a larger degree, although it depends itself on air temperature. The same picture was presented in research on different watersheds, including large lakes located outside city centres, e.g. Table Rock Lake (USA) (Dryden et al., 2006), Baikal Lake (Russia) (Drucker, Dutova, 2006), Pluβsee Lake (Germany) (Demuth et al., 1993), North Sea water (England) (Dore et al., 2000), estuaries of a river in California (USA) (Jiang, Paul, 1994), Norwegian fiords (Hernrothet al., 2002), Tampa Bay in Florida (USA) (Jiang et al., 2007) and water from Alzette River in Luxemburg (Fauvel et al., 2016), where high correlation between F-specific RNA phages, water temperature and their maximum level was recorded in colder, winter months. That data confirms our own data on F-RNA phages in the water of small lakes in Szczecin, in which their highest levels were recorded in winter (Śliwa-Dominiak et al. 2010, 2014)

Conclusions

1. Although the number of somatic and F-specific RNA and DNA phages in water samples from a municipal Rusałka Lake was 3,000 times lower than that of Contans Lake (Germany) (Hennes, Simon, 1995) and 8 times lower than that of Pluβsee Lake (Germany) (Demuth et al., 1993), our results show, without doubt, that phage presence also in small water reservoires, similarly to large reservoires, depends on and involves environmental conditions, including temperature of air and water (Cole et al., 2003; Dore et al., 2000; Fauvel et al., 2016; Hernroth et al., 2002; Jiang, Paul, 1994; Long, Sobsey, 2004; Olson et al., 2005; Simkova, Cervenka, 1981; Sinton et al., 1999; Skraber et al., 2004; Stewart et al., 2006; Śliwa-Dominiak et al., 2010, 2014). Our observations also confirm that, similarly to watersof large watersheds, there are more somatic than F-specific RNA and DNA phages, as the former are less susceptible to environmental conditions (Hernroth et al., 2002; Moce-Livina et al., 2005; Pusch et al., 2005; Sinton et al., 1999; Vergara et al., 2015). 2. Bacteriophages assay data, and particularly findings on F-specific RNA and DNA phages in small, municipal lake water, confirm their presence (Śliwa-Dominiaket al., 2010, 2014; Zupok et al., 2010) which seems to prove that water purity evaluation of aquous environments, including small lakes, should consider F-RNA phage presence in this environment, as it may affect the coliform count which determines water clarity.

52 Somatic and F-specific bacteriophages in waters of the small, municipal Rusałka Lake in Szczecin

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54 Somatic and F-specific bacteriophages in waters of the small, municipal Rusałka Lake in Szczecin

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Cite as: Pawlikowska-Warych, M., Czupryńska, P., Tokarz-Deptuła, B., Deptuła, W. (2019). Somatic and F-specific bacteriophages in watersof the small, municipal Rusałka Lake in Szczecin. Acta Biologica, 26, 45–55. DOI: 10.18276/ab.2019.26-05. #1#

#0#

Acta Biologica 26/2019 | www.wnus.edu.pl/ab | DOI: 10.18276/ab.2019.26-06 | strony 57–64

Changed species composition of naked amoebae in soils of forest-and-steppe zone of Ukraine

Marina Patsyuk

Zhytomyr Ivan Franko State University, Vel. Berdychivska st., 40, Zhytomyr, 10008 Ukraine

Corresponding author, e-mail: [email protected]

Keywords naked amoebae, morphotypes, soils, forest-and-steppe, Ukraine Abstract Twenty-three species of naked amoebae of 3 classes, 11 families and 16 genera were found in soils of the forest-and-steppe zone of Ukraine. The most common species were Vahlkampfia sp. (2), Vahlkampfia sp. (1), D. mycophaga, H. vermiformis, T. striata, R. platypodia, M. cantabrigiensis, Vexillifera sp., Cochliopodium sp. (1), Acanthamoeba sp. (1). The species with least occurrence were Polychaos sp., T. similis, T. terricola, M. viridis, Rhizamoeba sp. (1). Highest species diversity of naked amoebae was recorded for soils of forests and shrubs, least for soils of meadows. All of the found amoebae species belong to 12 morphotypes.

Zmiany składu gatunkowego ameb nagich w glebie w gradiencie siedlisk stepowych i leśnych Słowa kluczowe ameby nagie, morfotypy, gleba, gradient step-las, Ukraina Streszczenie Dwadzieścia trzy gatunki nagich ameb z 3 klas, 11 rodzin i 16 rodzajów znaleziono w glebach strefy leśno-stepowej Ukrainy. Najczęściej występującymi gatunkami były Vahlkampfia sp. (2), Vahlkampfia sp. (1), D. mycophaga, H. vermiformis, T. striata, R. platypodia, M. cantabrigiensis, Vexillifera sp., Cochliopodium sp. (1), Acanthamoeba sp. (1). Najwyższą różnorodność gatunkową nagich ameb odnotowano dla gleb lasów i krzewów, najmniejszą – dla gleb łąk. Wszystkie znalezione gatunki ameb należą do 12 morfotypów.

Introduction

The forest-and-steppe zone is the transitional area between the forest and steppe biocoenoses. The corresponding changes in composition structure of soil fauna can be easily observed there. According to current studies, there are both truly forest soils (gray and gray forest soils) and steppe soils (typical chernozems). Podzolic soils (dark gray podzols and podzolic chernozems), which form under forest and steppe biocoenoses, are a special case. In the forest-and-steppe zone, three provinces (Polisko-Prydnistrovskyi, Livoberezhno-Dnistrovskyi and Skhidno-Ukrainskyi) are recognized (Pankiv, 2017).

57 Marina Patsyuk

Naked amoebae are the usual components of soil, freshwater and seawater faunas. In Ukraine, these protists have been studied only in fresh waters (Patsyuk, Dovgal, 2012; Patsyuk, 2014, 2016a, 2018a). Our work aimed to analyze the changes in species composition of amoebae in soils of forest-and-steppe biocoenoses of Ukraine.

Materials and methods

Sampling was conducted in 2018–2019, and altogether 138 samples were collected in 12 locations (Figure 1). Soil cuts for protozoological analysis were established at study areas. Samples were taken from the surface soil horizon (0–5 cm). On each area, three pairs of samples were taken.

Figure 1. Sampling locations (Ukraine)

Soil cover was represented by chernozems characterized as intermediate between the dark gray forest soils to podzolic chernozems. Soil acidity was measured with laboratory pH-meter 150-М. The soil reaction varied from weakly acid (6.3) to neutral (7.2). Soil temperature was determined on the depth of down to 5 cm using soil thermometer. Samples was mostly taken during warm season, temperature of studied soils was in average +17°С. The amoebae were multiplied and maintained according to Page (1991) in laboratory conditions at +20°С. Our data are insufficient to make any conclusions about the abundanceof amoebae, thus we analyzed the frequency of finding the protists in soils of the forest-and-steppe zone of Ukraine. Cluster analysis was used to classify the protist communities. All calculations were conducted using the software program packet Past 1.18 (Hammer et al., 2001).

58 Changed species composition of naked amoebae in soils of forest-and-steppe zone of Ukraine

Results and discussion

The species composition of naked amoebae was studied in soils of the forest-and-steppe zone of Ukraine on 12 study plots. The plots were chosen to be comparatively distant from each other and with various plant associations of the forest-and-steppe ecotone, showing different stages of over-forestation of steppe (Mazei, Embulayeva, Trulova, 2013). Four plots were chosen at meadow steppe areas. The most common plant species included Elytrigia repens, Acuillea nobilis, Eringium campestre, Artemisia absinthium, Plantago major, Hieracium piloselioides, Artemisia absinthium, Agrimonia eupaforia, Berteroa incana, Polygonatum officinale etc. Four other plots were chosen under shrubs. The most prevalent plant species were Urtica dioisa, Sambucus nigra, Sorbus aucuparia, etc. And finally, four plots were established in forest areas. The most common trees were Quercus robur, Carpinus betulus, Betula pendula, Pinus sylvestris, Corylus avellana, Tilia cordata and other species. In the studied soils, 23 species of naked amoebae were found. These are listed below, according to the system in (Smirnov, Chao, Nassonova, Cavalier-Smith, 2011; Bass et al., 2009).

Class Tubulinea Smirnov et al., 2005 Order Tubulinida Smirnov et al., 2005 Family Amoebidae Ehrenberg, 1838 Genus Polychaos Schaeffer, 1926 Polychaos sp. Genus Deuteramoeba Page, 1987 Deuteramoeba mycophaga Page, 1988 Family Hartmannellidae (Volkonsky, 1931) Page, 1974 Genus Saccamoeba Frenzel, 1892 Saccamoeba stagnicola Page, 1974 Genus Hartmannella Page, 1974 Hartmannella vermiformis Page, 1967 Genus Cashia Page, 1974 Cashia limacoides Page, 1974 Order Leptomyxida Pussard et Pons, 1976 Family Leptomyxidae Pussard et Pons, 1976 Genus Rhizamoeba Page, 1972 Rhizamoeba sp. (1)

Class Discosea Cavalier-Smith et al., 2004 Subclass Flabellinia Smirnov et al., 2005 Order Dactylopodida Smirnov et al., 2005 Family Paramoebidae Poche, 1913 Genus Korotnevella Page, 1981 Korotnevella sp. (1) Family Vexilliferidae Page, 1987 Genus Vexillifera Schaeffer, 1926 Vexillifera sp.

59 Marina Patsyuk

Order Vannellida Smirnov et al., 2005 Family Vannellidae Bovee, 1970 Vannella sp. Genus Ripella Smirnov, Nassonova, Chao et Cavalier-Smith, 2007 Ripella platypodia Smirnov, Nassonova, Chao et Cavalier-Smith, 2007 Order Himatismenida Page, 1987 Suborder Tectiferina Smirnov, Nassonova, Chao et Cavalier-Smith, 2011 Family Cochliopodiidae De Saedeleer, 1934 Genus Cochliopodium Hertwig et Lesser, 1874 Cochliopodium sp. (1) Subclass Longamoebia Smirnov, Nassonova, Chao et Cavalier-Smith, 2011 Order Dermamoebida Cavalier-Smith et al., 2004 Family Mayorellidae Schaeffer, 1926 Genus Mayorella Schaeffer, 1926 Mayorella viridis Leidy, 1874 Mayorella cantabrigiensis Page, 1983 Mayorella sp. Order Thecamoebida Smirnov, Nassonova, Chao et Cavalier-Smith, 2011 Family Thecamoebidae Schaeffer, 1926 Genus Thecamoeba Fromentel, 1874 Thecamoeba striata Penard, 1890 Thecamoeba terricola (Greef, 1866) Lepsi, 1960 Thecamoeba similis Lepsi, 1960 Genus Stenamoeba Smirnov et al., 2007 Stenamoeba stenopodia (Page, 1969) Smirnov et al., 2007 Order Centramoebida Rogerson and Patterson, 2002 Family Acanthamoebidae Sawyer and Griffin, 1975 Genus Acanthamoeba Volkonsky, 1931 Acanthamoeba sp. (1)

Class Heterolobosea Page et Blanton, 1985 Family Vahlkampfiidae Jollos, 1917 Genus Vahlkampfia Chatton et Lalung-Bonnaire, 1912 Vahlkampfia sp. (1) Vahlkampfia sp. (2) Genus Willaertia De Jonckheere, Dive, Pussard et Vickerman, 1984 Willaertia sp. Genus Naegleria Alexeieff, 1912 Naegleria gruberi Schardinger, 1899

The most common species was Vahlkampfia sp. (2). It was also one of the dominant species in almost all local communities (Table 1). Vahlkampfia sp. (1), D. mycophaga, H. vermiformis, T. striata, R. platypodia, M. cantabrigiensis, Vexillifera sp., Cochliopodium sp. (1), Acanthamoeba sp. (1) (in total, 43.5% of all found species) were present in more than in 50% of biotopes. The representatives of class Disciosea were more prevalent (13 species, 56.52% of all

60 Changed species composition of naked amoebae in soils of forest-and-steppe zone of Ukraine identified species). Class Tubulinea was represented by six species (26.08%), and four species of class Heterolobosea were found (17.39%) (Figure 2).

Heterolobosea 17.39%

Discosea 56.52% Tubulinea 26.08%

Figure 2. Distribution of naked amoebae in soils of forest-and-steppe zone of Ukraine

By the species structure, the amoebae communities varied (Figure 3). The most specific communities were formed under shrub phytocoenoses. In those, the most frequent species were Vahlkampfia sp. (2), C. limacoides, and Mayorella sp. In all other biotopes, the most frequent species were Vahlkampfia sp. (1), H. vermiformis, Vannella sp., Vexillifera sp., Cochliopodium sp. (1), and Acanthamoeba sp. (1). The frequency of finding those species in samples was higher than 65%, and they made up 26% of all identified species. All of the most frequently found amoebae were the core of communities in several biotopes, determining the biocoenotic similarity of protists. However, the list of most common species was different in each community. Thus the variability of communities was shaped by recombining a small number of species.

Figure 3. Results of classification of naked amoebae communities from different biotopes by the species composition (nodes of the dendrogram show support values in percent, bootstrap 1,000)

61 Marina Patsyuk

By the species composition (Figure 4) the protist communities divided into two groups: from forest phytocoenoses, and from steppe and shrub biocoenoses. Five of the 23 found species were not found in soils of meadows and shrubs (Polychaos sp., M. viridis, T. terricola, T. similis, Korotnevella sp. (1)), four species were absent in meadow soils (D. mycophaga, S. stagnicola, C. limacoides, Willaertia sp.), one species in soils under shrubs (Rhizamoeba sp. (1)), one species was not found in soils of meadows and forests (Mayorella sp.) (Table 1).

Table 1. Species composition of naked amoebae in community

Biotope 2 3 4

No. Taxon 1 2 3 4 1 2 3 4 Meadow steppe 1 Meadow steppe Meadow steppe Meadow steppe Shrub Shrub Shrub Shrub Forest Forest Forest Forest 1. Vahlkampfia sp. (1) + + + + + + – – + + – + 2. Vahlkampfia sp. (2) + + + + + + + + + + + + 3. N. gruberi – – + – – + + – + – – – 4. Willaertia sp. – – – – – – + – – + + – 5. Polychaos sp. – – – – – – – – – + – + 6. D. mycophaga – – – – – + + – + + + + 7. S. stagnicola – – – – – + – – + + + + 8. H. vermiformis + + + + + – – + – – + + 9. C. limacoides – – – – + + + + – + – – 10. Rhizamoeba sp. (1) – – – + – – – – + + + – 11. Korotnevella sp. (1) – – – – – – – – + + + + 12. Vexillifera sp. + – + + + – – + + + + – 13. Vannella sp. – + + + – + – – + + + + 14. R. platypodia – + – + + – + – – + + + 15. Cochliopodium sp. (1) + – + – + + – – + + + – 16. M. viridis – – – – – – – – – – + – 17. M. cantabrigiensis + + – – + – + – – + + + 18. Mayorella sp. – – – – + + + + – – – – 19. T. striata – + – + – – + + + + + – 20. T. terricola – – – – – – – – – + + – 21. T. similis – – – – – – – – + – + – 22. S. stenopodia + + – – + + – – – + – – 23. Acanthamoeba sp. (1) + – – + + + – – + + – + 8 8 7 9 11 11 9 6 13 18 16 11 Total 13 17 22

In soils under shrubs, Vahlkampfia sp. (2), C. limacoides, and Mayorella sp. were the most common taxa, making up 17.6% of all identified species; the least common were Willaertia sp., S. stagnicola, Vannella sp. (17.6% of all noted species). Vahlkampfia sp. (1), N. gruberi, D. mycophaga, H. vermiformis, T. striata, R. platypodia, S. stenopodia, M. cantabrigiensis, Vexillifera

62 Changed species composition of naked amoebae in soils of forest-and-steppe zone of Ukraine sp., Cochliopodium sp. (1), and Acanthamoeba sp. (1) were recorded with the average frequency (64% of all amoeba found in soils under shrubs). In forest phytocoenoses, the most characteristic species were Vahlkampfia sp. (2), D. mycophaga, S. stagnicola, Vannella sp., and Korotnevella sp. (1) (23% of all found species). Four (18%) of 22 amoeba species were the least common: N. gruberi, C. limacoides, S. stenopodia, M. viridis. Finally, Vahlkampfia sp. (1), Polychaos sp., H. vermiformis, T. striata, T. terricola, T. similis, R. platypodia, M. cantabrigiensis, Vexillifera sp., Rhizamoeba sp., Cochliopodium sp. (1), and Acanthamoeba sp. (1) that made up 59% of all found amoebas, were found with average frequency. It should be noted that most of the identified species can be considered eurybiont because they can be found both in soils and in aquatic biotopes (Patsyuk, Dovgal, 2012; Patsyuk, 2014, 2016a, b, 2018a, b). It should be noted that the least numerous species in soils of the forest-and-steppe biocoenoses of Ukraine were Polychaos sp. (15%), T. similis (5%), T. terricola (7.5%), M. viridis (0.8%), and Rhizamoeba sp. (1) (12%). In soils of the forest-and-steppe biogeocoenoses of Ukraine, we found amoebae of 12 morphotypes: eruptive (Vahlkampfia sp. (1), Vahlkampfia sp. (2), N. gruberi, Willaertia sp.), polytactic (Polychaos sp., D. mycophaga), monotactic (S. stagnicola, H. vermiformis, C. limacoides), lens-like (Cochliopodium sp. (1)), striate (T. striata, T. similis), rugose (T. terricola), fan-shaped (Vannella sp., R. platypodia), mayorellian (M. viridis, M. cantabrigiensis, Mayorella sp.), dactylopodial (Korotnevella sp. (1), Vexillifera sp.), acanthopodial (Acanthamoeba sp. (1)), branched (Rhizamoeba sp. (1)), and lingulate (S. stenopodia). Amoebae of all morphotypes were recorded in forest soils, and of 10 morphotypes in meadow soils and under shrubs (where amoebae of polytactic and rugose, and of rugose and branched morphotypes were absent, respectively). In more than half studied biotopes we have found species of eruptive, polytactic, monotactic, mayorellian, dactylopodial, lens-like, striate, lanceolate, fan-shaped, and acanthopodial morphotypes, which is two thirds of all known morphotypes. Thus, we’ve identified 23 speciesof naked amoebae of 12 morphotypes in soils of the forest- and-steppe zone of Ukraine. The highest number of species was characteristic of forest soils (22 species) and soils under shrubs (17 species), which are 96% and 74% of all identified species. The lowest number of species was found in meadow soils (13 species, 56% of all found species). Possibly, the high diversity of amoebae in soils of forests and under shrubs of the forest-and-steppe zone of Ukraine strongly depends on the humidity and type of soil, a hypothesis which will be tested in detail in our further studies.

References

Bass, D., Chao, E., Nikolaev, S., Yabuki, A., Ishida, K., Berney, C., Pakzad, U., Wylezich, C., Cavalier- -Smith, T. (2009). Phylogeny of Novel Naked Filose and Reticulose Cercozoa: Granofilosea cl. n. and Proteomyxidea Revised. Protist, 160, 75–109. Hammer, Ø., Harper, D.A.T., Ryan, P.D. (2001). PAST: Palaeontological statistics software package for education and data analysis. Palaeontol. electronica, 4 (1), 1–9. Mazei, Yu., Embulayeva, E., Trulova, A. (2013). Terricolous testate amoebae in forest-steppe ecosystems (on the data of natural reserve «Volga region forest-steppe»). University Proceedings Volga Region, Natural Sciences, 2 (2), 5–26 [in Russian]. Page, F.C., Siemensma, F.J. (1991). Nackte Rhizopoda und Heliozoea (Protozoenfauna Band 2). Stuttgart– New York: Gustav Fischer Verlag.

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Pankiv, Z.P. (2017). Soils of Ukraine: a study book. Lviv, Ivan Franko State University, 112 [in Ukraine]. Patcyuk, M.K., Dovgal, I.V. (2012). Biotopic distribution of naked amoebes (Protista) in Ukrainian Polissya area. Vestnik zoologii, 46 (4), 355–360. Patsyuk, M.K. (2014). Morphotypes in Naked Amoebas (Protista): Distribution in Water Bodies of Zhy- tomyr and Volyn Polissia (Ukraine) and Possible Ecological Significance. Vestnik zoologii, 48 (6), 547–552. Patsyuk, M.K. (2016a). Seasonal changes in the species composition of naked amoebas (Amoebina) of the Teterev river (the Town of Zhitomir). Hydrobiological Jornal, 52 (4), 55–62. Patsyuk, M.K. (2016b). New Finds of Naked Amoebae (Protista) in Water Reservoirs of Ukraine. Vestnik zoologii, 50 (4), 291–300. Patsyuk, M.K. (2018a). Peculiarities of the Spatial Distribution of Naked Amoebas in Sandy Bottom Sedi- ments of a Small River. Hydrobiological Jornal, 54 (5), 102–111. Patsyuk, M. (2018b). Species Composition And Distribution Of Naked Amoebae In The Water Bodies of Lviv Region. Visnyk of the Lviv University, Series Biology, 79, 141–149. Smirnov, A., Chao, E., Nassonova, E., Cavalier-Smith, T. (2011). A Revised Classification of Naked Lobose Amoebae (Amoebozoa: Lobosa). Protist, 162, 545–570.

Cite as: Patsyuk, M. (2019). Changed species composition of naked amoebae in soils of forest-and-steppe zone of Ukraine. Acta Biologica, 26, 57–64. DOI: 10.18276/ab.2019.26-06. #1#

64 #0#

Acta Biologica 26/2019 | www.wnus.edu.pl/ab | DOI: 10.18276/ab.2019.26-07 | strony 65–81

Tetranychus urticae changes its oviposition pattern in the presence of the predatory mites, Phytoseiulus persimilis and Typhlodromus bagdasarjani

Mona Moghadasi, Azadeh Zahedi Golpayegani, Alireza Saboori, Hossein Allahyari, Hamideh Dehghani Tafti

Department of Plant Protection, Faculty of Agriculture, University of Tehran, Karaj, Iran

Corresponding author, e-mails: [email protected], [email protected], [email protected], [email protected]

Keywords oviposition, spider mite, predation, parental care, Phytoseiidae Abstract Oviposition behaviors in herbivorous mites are affected by several factorsi.e. food availability for juveniles and reduced predation risks. We used the twospotted spider mite, Tetranychus urticae Koch (Tetranychidae) to find out whether the previous presenceof specialist/ generalist phytoseiid predator individuals, Phytoseiulus persimilis Athias-Henriot/ Typhlodromus bagdasarjani Wainstein & Arutunjan (here, direct effect) or their previous odour perception by prey (here, indirect effect) would affectT. urticae oviposition strategies. Tetranychus urticae female individuals were placed on a leaf disc in a plastic container with predators either on the same disc (direct presence of predator) or on the second disc (receiving odours related to a predator) in the same container. Getting experienced, the prey individuals transferred to the oviposition container to their oviposition pattern parameters get recorded. The ovipositing T. urticae were monitored in two experimental situations: 1. Receiving odours related to the predator- prey interaction from the second leaf disc in the same oviposition container during their oviposition period, and 2. Receiving no odour. Our results showed that when T. urticae females perceived the predator presence in their first container (with either predator species, both direct and indirect effect), they reduced their total egg distances, oviposition rates and oviposition periods significantly regardlessof receiving odours related to prey-predator interactions during experiment. Receiving odours during oviposition, T. urticae females decreased their pairwise egg distances in at least 4 and at most 6 pairs of eggs, while when odours were absent during oviposition, the distances decreased in at least 2 and at most 3 pairs of eggs. The direct presence of P. persimilis reduced the prey oviposition period significantly more than that when T. bagdasarjani was present. The spider mites oviposition rate reduction was obviousely more than that in the presence of T. bagdasarjani. The different effectsof predator species on T. urticae egg distances were discussed.

65 Mona Moghadasi, Azadeh Zahedi Golpayegani, Alireza Saboori, Hossein Allahyari, Hamideh Dehghani Tafti

Zmiana wzoru składania jaj przez Tetranychus urticae w obecności drapieżnych roztoczy, Phytoseiulus persimilis i Typhlodromus bagdasarjani Słowa kluczowe składanie jaj, przędziorki, drapieżnictwo, opieka rodzicielska, Phytoseiidae Streszczenie Na zachowania związane ze składaniem jaj roztoczy roślinożernych wpływa kilka czynników między innymi dostępność pokarmu dla młodych osobników i zmniejszone ryzyko drapieżnic- twa. Użyliśmy przędziorka Tetranychus urticae Koch (Tetranychidae), aby dowiedzieć się, czy obecność drapieżnika określonego typu (wyspecjalizowany w drapieżnictwie na Tetranychus urticae przedstawiciel drapieżnych dobroczynkowatych, Phytoseiulus persimilis Athias- Henriot i niewyspecjalizowany Typhlodromus bagdasarjani Wainstein & Arutunjan) wpływają na strategie składania jaj T. urticae. Następnie powtórzyliśmy eksperyment używając zapachu poprzednich ofiar tych drapieżników. Samice Tetranychus urticae umieszczono na krążku z liści w plastikowym pojemniku z drapieżnikami na tym samym krążku (bezpośrednia obec- ność drapieżnika) lub na drugim krążku (odbierającym zapachy związane z drapieżnikiem) w tym samym pojemniku. Po doświadczeniu, ofiary przenoszono do pojemnika, w którym samice składały jaja. Parametry związane ze składaniem jaj T. urticae monitorowano w dwóch grupach doświadczalnych: grupa eksperymentalna otrzymywała zapach związany z interakcją drapieżnik-ofiara, natomiast grupa kontrolna nie otrzymywała zapachu. Nasze wyniki wyka- zały, że gdy samice T. urticae dostrzegły obecność drapieżnika lub jego zapach (w przypadku obu gatunków drapieżników), znacznie zmniejszyły liczbę składanych jaj i odległości między składanymi jajami, a ponadto skracały okres składania jaj, niezależnie od aplikowania za- pachów związanych z interakcją między drapieżnikiem i ofiarą. Jednak aplikacja zapachu podczas składania jaj powodowała zmniejszenie odległości między składanymi jajami w od 4 do 6 par kolejno składanych jaj. W grupie kontrolnej, odległości zmniejszyły się w od 2 lub 3 parach kolejno składanych jaj. Bezpośrednia obecność P. persimilis skróciła okres składania jaj przez ofiarę znacznie bardziej niż obecność T. bagdasarjani. Zmniejszenie szybkości składania jaj przez przędziorki było wyraźnie większe niż w obecności T. bagdasarjani.

Introduction

Animals with a range of specialist and non-specialist predators are confronted with many time and energy costing risks during their foraging behavior. The behavioral decisions made under the risk of predation are more imperative than those made to detect food (Lima, Dill, 1990). Many organisms show defensive or evasive behaviors when they receive chemical information related to their predators (direct cues of predation risk) or from the conspecifics injured by those predators (indirect cues of predation risk). The prey different reactions in these situations depend on several factors i.e. how familiar the prey is with the alerting cues, the predator risk level and the prey strategies to avoid predator (Grostal, Dicke, 1999). These crucial traits that influence the prey behavior, distribution and whole fitness, could also lead to a range of new indirect interactions in the food web (Abrams, 1996). Failing to detect a high-risk predator increases the encounter rate of prey with predator which might have important implications on its survival and reproductive success. Several studies have demonstrated that once a predator has been detected, prey changes its behavior so that decrease its vulnerability. The crayfish,Paranephrops zealandicus (Koura) reduced its stationary and walking behavior when it received chemicals related to eel (Anguilla dieffenbachia) (Shave, Townsend, Crowl, 1994). Daphnia magna Straus, respond to chemicals related to its injured conspecifics (crushed Daphnia) by formation of aggregation and vertical distribution towards the bottom (Pijanowska, 1997). The fruit flies (Rhagoletis basiola (Osten Sacken), Tephritidae) delayed their oviposition when received cues related to their egg parasitoid (Hoffmeister, Roitberg, 1997). Dias

66 Tetranychus urticae changes its oviposition pattern in the presence of the predatory mites... et al. (2016) demonstrated that the response of prey to the cues related to dangerous and harmless predators could be different. Several studies have demonstrated that when a predator and its con- or hetero-specific competitor are present together, the former should adopt strategies in order to decrease the risk of its offspring being preyed upon. Montserrat et al. (2007) showed that females of Neoseiulus cucumeris (Oudemans) reduced oviposition by retaining eggs inside their body when exposed to Iphiseius degenerans (Berlese). Amblyseius swirskii (Athias-Henriot) could anticipate the possible counterattacks on behalf of Frankliniella occidentalis Pergande and avoided ovipositing near thrips (de Almedia, Janssen, 2013). I. degenerans oviposits away from their food source (pollen) when the predator of its eggs, F. occidentalis was present on the flower (Faraji, Janssen, van Rijn, Sabelis, 2000). Spider mites are known to show anti-predator responses through behaviors such as patch avoidance, induced diapause, reduction of oviposition, producing dense web and moving away from the patch which the predator is present in (Pallini, Janssen, Sabelis, 1999; Choh, Takabayashi, 2006; Kroon, Veenendaal, Bruin, Egas, Sabelis, 2008; Shimoda, Kishimoto, Takabayashi, Amano, Dicke, 2009; Lemos et al., 2010). Dittmann and Schausberger (2017) documented that the aggregated spider mites were less encountered by Phytoseiulus persimilis Athias- Henriot in comparison with the scattered ones. They suggested that more aggregation (inter-individual distance reduction) in T. urticae would lead to more efficacious anti-predator behaviours. Dias et al. (2016) reported that Tetranychus evansi Baker and Pritchard oviposited less and escaped more when their dangerous predator Phytoseiulus longipes Evans was present on the same patch. They concluded that spider mite antipredator behaviours varied with the level of the predator danger. Hackl and Schausberger (2014) were the first who demonstrated that experience and learning could modulate T. urticae anti-predator behaviours. They reported that previous predator experience would lead to a balanced response of T. urticae to P. persimilis cues in the shape of oviposition delay and activity reduction, but not reducing the oviposition rate. They also did not discuss whether the predator perception by T. urticae would lead to the prey oviposition pattern. Typhlodromus bagdasarjani Wainstein & Arutunjan is a generalist indigenous phytoseiid mite (Type III) with a wide distribution in the Middle East, especially in the orchards of Iran and reported from plants infested by tetranychids and eriophyids as well as insect pests such as thrips and whiteflies. Various plant exudates, as well as honydew, may serve as supplements which could increase its reproduction potential in food scarcity. T. bagdasarjani could be well adapted to warm climates (Farazmand, Fathipour, Kamali, 2013; Moghadasi, Saboori, Allahyari, Zahedi Golpayegani, 2013; McMurtry, De Moraes, Sourassou, 2013). Phytoseiulus persimilis Athias-Henriot is a specialist (Type I) commercialized predatory mite that is commonly used in ornamental greenhouses to control spider mites. This predatory mite has a short generation time with high fecundity and its post-larval stages are able to attack all stages of T. urticae (Moghadasi et al., 2013; Landeros, Guevara, Badii, Flores, Pamanes, 2004; Pizzol, Poncet, Hector, Ziegler, 2006). Here, we have investigated the direct and indirect effects of the predatory mites, P. persimilis and T. bagdasarjani on the oviposition rate, distances between eggs and oviposition period of T. urticae. Recording the direct effects, we put the predator female individuals (each species separately) on the same leaf disc which T. urticae was present. For indirect effects, no predator was introduced to the spider mite leaf disc, but the ovipositing spider mites received the odours related to interacing prey- predator from the nearby leaf disc in the same container. We tested whether continuing to receive odours during oviposition could also affect T. urticae oviposition behavior. We expected that spider mites should reduce the distances between eggs and increase

67 Mona Moghadasi, Azadeh Zahedi Golpayegani, Alireza Saboori, Hossein Allahyari, Hamideh Dehghani Tafti

their oviposition period when perceived the predator effects. The effect of predator species on the prey strategies is discussed.

Materials and Methods

Plants and mites Rose plants (Rosa hybrida cv. ‘blarodje’ (Rosaceae)) used in this study were grown in beds under commercial conditions (Cocopeat: Perlit; 60: 40%) in the greenhouse. Plants were pruned and planted in large plastic pots (25 cm top diameter, 40 cm depth and 20 cm bottom diameter) at 25 ±2°C, 65 ±10% RH and 16L: 8D hour photoperiod. Spider mites, T. urticae, were originally collected from infested lima bean cultures in the acarology laboratory at the Department of Plant Protection, Faculty of Agriculture, University of Tehran, Karaj, Iran. Spider mites were transferred onto detached rose leaves placed upside down on water-saturated cotton wool in transparent plastic containers (20 × 10 × 4 cm). The rearing containers were maintained in controlled conditions at 24 ±2°C, 60 ±5% RH, and 16L: 8D hour photoperiod, since three months prior to experiments. The predatory mites, T. bagdasarjani were collected from black mulberry trees at the campus of Tarbiat Modares University, Tehran, Iran. Phytoseiulus persimilis culture was initially obtained from laboratory stock culture reared at the Acarology laboratory at the Department of Plant Protection, Faculty of Agriculture, University of Tehran, Karaj, Iran. The predatory mites were kept on masses of detached rose leaves deposited upside down on a plastic sheet placed on a water saturated sponge. The plastic sheet was surrounded by napkin tapes which were put into the water from another side so that the predatory mites could drink water. The rearing units were kept at controlled conditions (25 ±1°C, 75 ±5% RH and 16L: 8D hour photoperiod) in a growth chamber. All experiments were carried out at 25–26°C, 75–80% RH, and 16L: 8D hour photoperiod in growth chamber.

General experimental conditions The experimental units consisted of fresh detached rose leaf discs (2 × 2 cm2) placed upside down on semi-divided water-saturated sponges in a porous (8 pore) transparent plastic container (Figure 1). Each pair of cubic sponges was adjusted in a way that the cubes were spaced 15 mm

Figure 1. The experimental unit

68 Tetranychus urticae changes its oviposition pattern in the presence of the predatory mites... from each other. Wet napkin strips were placed around the leaf margins in order to prevent the mites from escaping. Here, the preconditioning plastic container and the oviposition container were considered as the places for the spider mites to get experienced with odors and to check their oviposition behaviors respectively. Depending on the experiments, one of or both of the leaf discs in each container were used.

Experiments The oviposition pattern of T. urticae when experienced the predator presence (Direct effect) Quiescent T. urticae female deutonymphs were selected from the main culture and put on separate leaf discs. After 24 hours, adult same-aged males were introduced to the females for mating. Newly mated T. urticae females were selected randomly (Choh, Uefune, Takabayashi, 2010). Two same-aged predator females (4 days old, predation rate at pick, unpublished data) and 10 same-aged T. urticae mated females were introduced to a plastic container (one of the pairwise leaf discs) mentioned above. 24 hours after the predator-prey interactions, one alive T. urticae female was selected randomly and transferred to a new leaf disc singly in a separate container (namely oviposition container) as treatment. Tetranychus urticae mated females (same-aged with those in treatment) which had not experienced the predator presence (in their preconditioning container) were transferred to a new leaf disc (namely oviposition container) singly and considered as control. This experiment was conducted in two situations: 1) When T. urticae females did not receive the odours related to predator-prey interactions in their oviposition container (Figure 2), and 2) when T. urticae females received odours related to predators and prey interactions from the second leaf disc in the same (oviposition) container (Figure 3). The second leaf discs both in the preconditioning (experiencing) and the oviposition container was prepared by introducing three same-aged female predators along with 10 ovipositing T. urticae 24 hours prior to monitoring got started, so that the prey and predators had sufficient time to interact. We kept recording data until

Figure 2. The experimental unit for recording the oviposition behavior of T. urticae females when received no odours related to interacting prey and predators (Direct effect)

69 Mona Moghadasi, Azadeh Zahedi Golpayegani, Alireza Saboori, Hossein Allahyari, Hamideh Dehghani Tafti

the female spider mites died. Data were recorded for T. urticae as follows: a) Total number of eggs each female oviposited till its death, b) Pairwise and total egg distances by regular monitoring, c) The oviposition period of spider mite females. The oviposition priority of eggs was shown by different watercolor spots put beside them. Distances were measured by ruler under binocular microscope (Schausberger, 2005). Each experiment was replicated 20 times.

Figure 3. The experimental unit for recording the oviposition behavior of T. urticae females when received odours related to interacting prey and predators from the second leaf disc (Direct effect)

The oviposition pattern of T. urticae when experienced volatiles related to the predator (Indirect effect) Two same-aged predator females (4 days old, predation rate at the pick, unpublished data) and 10 same-aged T. urticae mated females were introduced into a plastic container (one of the pairwise leaf discs). The second leaf disc of the container was infested by 10 T. urticae mated females (without predators). As both discs were kept in the same container (mentioned above), the spider mite females of the second leaf disc received the odours related to the interactions among their conspecifics with the predators in the first leaf disc. After 24 hours, one T. urticae was selected from the second leaf disc randomly and transferred to a new leaf disc in a new (oviposition) container (treatment). Tetranychus urticae mated females (same-aged with those in treatment) which had just experienced the odours related to conspecifics interactions, were transferred to their oviposition container singly and considered as control. This experiment was conducted in two situations: 1) When T. urticae females did not receive the odours related to predator-prey interactions in their second (oviposition) container, and 2) when T. urticae female in the oviposition container (during oviposition) received odours related to predator-prey interactions from the second leaf disc of the same container. We kept recording data until the female spider mites died. Data were recorded for T. urticae as follows: a) The total number of eggs each female oviposited till its death, b) pairwise and total egg distances by regular monitoring (just for the first seven oviposited eggs as recording such data for the whole (30–40) eggs would be time- consuming), c) The oviposition period of spider mite females. The oviposition priority of eggs

70 Tetranychus urticae changes its oviposition pattern in the presence of the predatory mites... was shown by different watercolor spots put beside them. Distances were measured by ruler under binocular. Each experiment was replicated 20 times.

Data analysis The oviposition parameters of T. urticae in the presence of P. persimilis were recorded simultaneously with those in the presence of T. bagdasarjani. Independent sample-test was performed to compare the total number of eggs, oviposition period and pairwise and total egg distance between treatments as well as between each treatment and control using SPSS 16.

Results

The oviposition pattern of T. urticae when experienced the predator presence (Direct effect) Receiving no odours in the oviposition container during oviposition When T. urticae females experienced the predator (either P. persimilis or T. bagdasarjani) presence in their preconditioning container, they reduced pairwise distances between their first three eggs significantly (Tables 1 and 2, P < 0.01). The pairwise distances between their 4th to 7th eggs did not show any significant differenceP ( > 0.05). Tetranychus urticae total egg distances were significantly shorter when the females experienced the predator presence before starting oviposition (Table 1, P < 0.05) (Table 2, P < 0.01). Also, T. urticae females laid significant fewer eggs when they previously had perceived the predator presence (Tables 1 and 2, P < 0.01). The oviposition periods also decreased when the females experienced the predator presence prior to oviposition (Tables 1 and 2, P < 0.01).

Table 1. The total number of eggs, egg distances (mm) and oviposition period (day) of T. urticae females when experienced P. persimilis presence in their preconditioning container

Predator presence Control Source of variation P (Mean ± SE) (Mean ± SE) 1–2 7.7 ±0.73 11.8 ±1.18 0.005** 2–3 7.45 ±0.71 10.4 ±1.02 0.022** Pairwise egg 3–4 6.57 ±0.74 11.22 ±0.88 0.000** distances 4–5 7.72 ±0.75 8.02 ±1.04 0.816 5–6 8.4 ±0.42 7.95 ±0.84 0.641 6–7 8.27 ±0.42 8.17 ±0.65 0.898 Total egg distance (1–7) 46.12 ±2.65 57.58 ±3.31 0.010* Mean of egg distances 7.67 ±0.44 9.59 ±0.55 0.010* Total number of eggs 24.15 ±1.02 34.7 ±1.69 0.000** Oviposition period 10.45 ±0.11 11.65 ±0.13 0.000**

71 Mona Moghadasi, Azadeh Zahedi Golpayegani, Alireza Saboori, Hossein Allahyari, Hamideh Dehghani Tafti

Table 2. The total number of eggs, egg distances (mm) and oviposition period (day) of T. urticae females when experienced T. bagdasarjani presence in their preconditioning container

Predator presence Control Source of variation P (Mean ± SE) (Mean ± SE) 1–2 7.22 ±0.69 11.8 ±1.18 0.002** 2–3 6.82 ±0.82 10.4 ±1.02 0.009** Pairwise egg 3–4 7.45 ±0.69 11.22 ±0.88 0.002** distances 4–5 8.07 ±0.62 8.02 ±1.04 0.967 5–6 8.4 ±0.75 7.95 ±0.84 0.697 6–7 7 ±0.66 8.17 ±0.65 0.213 Total egg distance (1–7) 44.97 ±2.99 57.58 ±3.31 0.010* Mean of egg distances 7.49 ±0.49 9.59 ±0.55 0.008* Total number of eggs 25.55 ±1.38 34.7 ±1.69 0.000** Oviposition period 10.75 ±0.09 11.65 ±0.13 0.000**

The pairwise estimated distances from the 1st to the 7th T. urticae eggs did not differ significantly when they experienced either P. persimilis or T. bagdasarjani. This was also true for their total egg distances (Table 3, P < 0.05). Although the total number of T. urticae eggs was lower in the presence of P. persimilis in comparison with in the presence of T. bagdasarjani, no significant difference was observed between them P( < 0.05). The oviposition period of females also did not show any significant difference in the presenceof each of the predators (Table 3, P > 0.05).

Table 3. The total number of eggs, egg distances (mm) and oviposition period (day) of T. urticae females when experienced either P. persimilis or T. bagdasarjani presence in their preconditioning container

P. persimilis presence T. bagdasarjani presence Source of variation P (Mean ± SE) (Mean ± SE) 1–2 7.7 ±0.73 7.22 ±0.69 0.637 2–3 7.45 ±0.71 6.82 ±0.82 0.569 Pairwise egg 3–4 6.57 ±0.74 7.45 ±0.69 0.395 distances 4–5 7.72 ±0.75 8.07 ±0.62 0.721 5–6 8.4 ±0.42 8.4 ±0.75 1.000 6–7 8.27 ±0.42 7 ±0.66 0.113 Total egg distance (1–7) 46.12 ±2.65 44.97 ±2.99 0.775 Mean of egg distances 7.69 ±0.44 7.49 ±0.49 0.778 Total number of eggs 24.15 ±1.02 25.55 ±1.38 0.422 Oviposition period 10.45 ±0.11 10.75 ±0.09 0.055

Receiving predator-prey odours during oviposition In the treatments which T. urticae females not only had experienced the predator (P. persimilis) presence in their preconditioning container, but also received odours related to predator- prey interactions from the second leaf disc of the oviposition container (during oviposition), the pairwise distances between the 1st to the 7th eggs were decreased whereas the distance reduction between the 4th and 5th eggs was not significant (Table 4).Tetranychus urticae total egg distance,

72 Tetranychus urticae changes its oviposition pattern in the presence of the predatory mites... the total number of eggs and oviposition period were significantly reduced by receiving predator- prey odours during oviposition (Table 4, P < 0.01).

Table 4. The total number of eggs, egg distances (mm) and oviposition period (day) of T. urticae females when experienced P. persimilis in their proconditioning container and received predator-prey odours during oviposition

Predator presence Control Source of variation P (Mean ± SE) (Mean ± SE) 1–2 5 ±0.69 10.7 ±1.14 0.000** 2–3 6.4 ±0.66 10.27 ±0.91 0.001** Pairwise egg 3–4 5.5 ±0.93 10.97 ±0.93 0.000** distances 4–5 5.9 ±0.99 8.6 ±0.93 0.054 5–6 5.3 ±0.53 8.7 ±0.67 0.000** 6–7 6.8 ±0.58 8.45 ±0.52 0.041* Total egg distance (1–7) 34.9 ±3.43 57.7 ±3.45 0.000** Mean of egg distances 5.82 ±0.57 9.62 ±0.57 0.000** Total number of eggs 19.4 ±0.59 34.45 ±1.7 0.000** Oviposition period 10.6 ±0.15 11.7 ±0.10 0.000**

Receiving odours related to T. bagdasarjani-T. urticae interactions from the second leaf disc of the oviposition container (during oviposition), T. urticae females reduced the pairwise distances between their 1st to 4th and 6th to 7th eggs significantly when they experienced T. bagdasarjani in their preconditioning container (Table 5). Although the distances between the eggs 4th to 5th and 5th to 6th were reduced in comparison with control, the differences were not significant. Tetranychus urticae total egg distance, the total number of eggs and oviposition period were significantly reduced by receiving predator-prey odours during oviposition (Table 5, P < 0.01).

Table 5. The total number of eggs, egg distances (mm) and oviposition period (day) of T. urticae females when experienced T. bagdasarjani in their preconditioning container and received predator-prey odours during oviposition

Predator presence Control Source of variation P (Mean ± SE) (Mean ± SE) 1–2 5.95 ±1.04 10.7 ±1.14 0.004** 2–3 6.55 ±1.04 10.27 ±0.91 0.011* Pairwise egg 3–4 7.12 ±0.96 10.97 ±0.93 0.006** distances 4–5 6.72 ±0.87 8.6 ±0.93 0.148 5–6 7.95 ±0.92 8.7 ±0.67 0.514 6–7 6.07 ±0.70 8.45 ±0.52 0.010* Total egg distance (1–7) 40.37 ±3.55 57.7 ±3.45 0.001** Mean of egg distances 6.73 ±0.59 9.62 ±0.57 0.001** Total number of eggs 24.55 ±1.7 34.45 ±1.7 0.000** Oviposition period 10.75 ±0.12 11.7 ±0.10 0.000**

The pairwise estimated distances from the 1st to the 7th T. urticae eggs did not differ significantly when they experienced either P. persimilis or T. bagdasarjani. This was also true for

73 Mona Moghadasi, Azadeh Zahedi Golpayegani, Alireza Saboori, Hossein Allahyari, Hamideh Dehghani Tafti

their total egg distances (Table 6, P < 0.05). The total number of T. urticae eggs was lower in the presence of P. persimilis (P < 0.05). The oviposition period of females also did not show any significant difference in the presenceof each of the predators (Table 6, P > 0.05).

Table 6. The total number of eggs, egg distances (mm) and oviposition period (day) of T. urticae females when experienced either P. persimilis or T. bagdasarjani presence in their preconditioning container and received the same predator-prey odours during oviposition

P. persimilis presence T. bagdasarjani presence Source of variation P (Mean ± SE) (Mean ± SE) 1–2 5 ±0.69 5.95 ±1.04 0.454 2–3 6.4 ±0.66 6.55 ±1.04 0.904 Pairwise egg 3–4 5.5 ±0.93 7.12 ±0.96 0.232 distances 4–5 5.9 ±0.99 6.72 ±0.87 0.535 5–6 5.3 ±0.53 7.95 ±0.92 0.017* 6–7 6.8 ±0.58 6.07 ±0.70 0.430 Total egg distance (1–7) 34.9 ±3.43 40.37 ±3.55 0.274 Mean of egg distances 5.82 ±0.57 6.73 ±0.59 0.274 Total number of eggs 19.4 ±0.59 24.55 ±1.7 0.007* Oviposition period 10.6 ±0.15 10.75 ±0.12 0.448

The oviposition pattern of T. urticae when received odours related to the predator- prey interactions in their preconditioning container (Indirect effect) Receiving no odours in the oviposition container during oviposition When T. urticae females experienced the presence of P. persimilis in their preconditioning container, they reduced their pairwise distances between their 1st and 2nd (P < 0.05) and 3rd and 4th (P < 0.01) eggs significantly (Table 7).The pairwise distances between their 4th to 7th eggs did not show any significant difference (P > 0.05) and the distances between 2nd and 3rd eggs did likewise. Tetranychus urticae total egg distance was significantly shorter when the females had experienced P. persimilis presence prior to oviposition (Table 7, P < 0.05), also T. urticae females preferred to lay significant fewer eggs when they previously had perceived the predator presence (Table 7, P < 0.01). The oviposition period also decreased when the female spider mites had experienced P. persimilis prior to oviposition (Table 7, P < 0.01). When T. urticae females experienced the presence of T. bagdasarjani in their preconditioning container, they reduced their pairwise distances between their 1st to 3rd eggs significantly (Table 8, P < 0.05). The pairwise distances between their 4th to 7th eggs did not show any significant difference P( > 0.05). Tetranychus urticae total egg distance was significantly shorter when the females had experienced T. bagdasarjani presence prior to oviposition (Table 8, P < 0.05), also T. urticae females preferred to lay significant fewer eggs when they previously had perceived the predator presence (Table 8, P < 0.01). The oviposition period also decreased when the female spider mites had experienced T. bagdasarjani prior to oviposition (Table 8, P < 0.01).

74 Tetranychus urticae changes its oviposition pattern in the presence of the predatory mites...

Table 7. The total number of eggs, egg distances (mm) and oviposition period (day) of T. urticae females when received odours related to predator (P. persimilis)-prey interactions in their preconditioning container

Predator presence Control Source of variation P (Mean ± SE) (Mean ± SE) 1–2 8.2 ±0.82 11.2 ±1.02 0.028* 2–3 7.82 ±0.79 10.3 ±1.04 0.066 Pairwise egg 3–4 6.9 ±0.88 11.45 ±0.98 0.001* distances 4–5 8.15 ±0.79 9.47 ±1.01 0.311 5–6 9.07 ±0.51 7.47 ±0.81 0.102 6–7 8.42 ±0.59 8.97 ±0.80 0.583 Total egg distance (1–7) 48.57 ±2.68 58.87 ±3.41 0.023* Mean of egg distances 8.09 ±0.45 9.81 ±0.57 0.023* Total number of eggs 22.7 ±0.75 35.35 ±1.68 0.000** Oviposition period 10.45 ±0.15 12.2 ±0.18 0.000**

Table 8. The total number of eggs, egg distances (mm) and oviposition period (day) of T. urticae females when received odours related to predator (T. bagdasarjani)-prey interactions in their preconditioning container

Predator presence Control Source of variation P (Mean ± SE) (Mean ± SE) 1–2 8.1 ±0.71 11.2 ±1.02 0.017* 2–3 7 ±0.77 10.3 ±1.04 0.015* Pairwise egg 3–4 8.27 ±0.87 11.45 ±0.98 0.021* distances 4–5 9.12 ±0.79 9.47 ±1.01 0.788 5–6 9.07 ±0.84 7.47 ±0.81 0.178 6–7 7.35 ±0.79 8.97 ±0.80 0.159 Total egg distance (1–7) 48.92 ±2.69 58.87 ±3.41 0.028* Mean of egg distances 8.15 ±0.45 9.81 ±0.57 0.028* Total number of eggs 26.5 ±1.43 35.35 ±1.68 0.000** Oviposition period 10.6 ±0.13 12.2 ±0.18 0.000**

Table 9. The total number of eggs, egg distances (mm) and oviposition period (day) of T. urticae females when experienced odours related to predator (P. persimilis /T. bagdasarjani)-prey interactions in their procontioning container

P. persimilis presence T. bagdasarjani presence Source of variation P (Mean ± SE) (Mean ± SE) 1–2 8.2 ±0.82 8.1 ±0.71 0.927 2–3 7.82 ±0.79 7 ±0.77 0.461 Pairwise egg 3–4 6.9 ±0.88 8.27 ±0.87 0.273 distances 4–5 8.15 ±0.79 9.12 ±0.79 0.392 5–6 9.07 ±0.51 9.07 ±0.84 1.000 6–7 8.42 ±0.59 7.35 ±0.79 0.284 Total egg distance (1–7) 48.57 ±2.68 48.92 ±2.69 0.927 Mean of egg distances 8.09 ±0.45 8.15 ±0.45 0.926 Total number of eggs 22.7 ±0.75 26.5 ±1.43 0.024* Oviposition period 10.45 ±0.15 10.6 ±0.13 0.466

75 Mona Moghadasi, Azadeh Zahedi Golpayegani, Alireza Saboori, Hossein Allahyari, Hamideh Dehghani Tafti

The pairwise estimated distances from the 1st to the 7th T. urticae eggs did not differ significantly between the treatments which received odours related to P. persimilis or T. bagdasarjani. It was also happened for their total egg distances (Table 9, P < 0.05). The total number of T. urticae eggs was lower in the presence of P. persimilis (P < 0.05). The oviposition period of females also did not show any significant difference in the presenceof each of the predators (Table 9, P > 0.05).

Receiving predator-prey odours during oviposition In the treatments which T. urticae females not only had experienced the predator (P. persimilis) presence in their preconditioning container but also received odours related to predator-prey interactions from the second leaf disc of the oviposition container (during oviposition), the pairwise distances between the 1st to the 7th eggs were decreased significantly (Table 10).Tetranychus urticae total egg distance, the total number of eggs and oviposition period were significantly reduced by receiving predator-prey odours during oviposition (Table 10, P < 0.01).

Table 10. The total number of eggs, egg distances (mm) and oviposition period (day) of T. urticae females when received odours related to predator (P. persimilis)-prey interactions in their preconditioning and oviposition containers

Predator presence Control Source of variation P (Mean ± SE) (Mean ± SE) 1–2 5.17 ±0.71 10.15 ±1.34 0.002** 2–3 6.05 ±0.61 10.32 ±0.93 0.000** Pairwise egg 3–4 4.85 ±0.83 10.7 ±1.11 0.000** distances 4–5 5.87 ±0.80 8.46 ±0.78 0.026* 5–6 4.72 ±0.57 8.22 ±0.69 0.000** 6–7 6.95 ±0.57 8.57 ±0.55 0.049* Total egg distance (1–7) 33.62 ±2.73 56.44 ±3.16 0.000** Mean of egg distances 5.6 ±0.45 9.41 ±0.53 0.000** Total number of eggs 19.2 ±0.71 33.55 ±1.77 0.000** Oviposition period 10.15 ±0.21 11.4 ±0.15 0.000**

Receiving odours related to T. bagdasarjani-T. urticae interactions during oviposition, T. urticae females reduced the pairwise distances between their 1st to 4th and 6th to 7th eggs significantly when they experienced T. bagdasarjani in their preconditioning container (Table 11). Although the distances between the eggs 4th to 5th and 5th to 6th were reduced in comparison with control, the differences were not significant. Tetranychus urticae total egg distance, the total number of eggs and oviposition period were significantly reduced by receiving predator-prey odours during oviposition (Table 11, P < 0.01). The pairwise estimated distances from the 1st to the 7th T. urticae eggs did not differ significantly (P > 0.05) except the distances between the 5th and 6th eggs (P < 0.01) between the treatments which received odours related to P. persimilis and T. bagdasarjani. It was also happened for their total egg distances (Table 12, P > 0.05). The total number of T. urticae eggs was lower in the presence of P. persimilis (P < 0.05). The oviposition period of females also did not show any significant difference in the presenceof each of the predators (Table 12, P > 0.05).

76 Tetranychus urticae changes its oviposition pattern in the presence of the predatory mites...

Table 11. The total number of eggs, egg distances (mm) and oviposition period (day) of T. urticae females when received odours related to predator (T. bagdasarjani)-prey interactions in their preconditioning and oviposition containers

Predator presence Control Source of variation P (Mean ± SE) (Mean ± SE) 1–2 5.75 ±1.05 10.15 ±1.34 0.014* 2–3 6.62 ±1.07 10.32 ±0.93 0.013* Pairwise egg 3–4 6.57 ±1.09 10.7 ±1.11 0.012* distances 4–5 6.57 ±1.07 8.46 ±0.78 0.162 5–6 7.67 ±0.89 8.22 ±0.69 0.629 6–7 6.47 ±0.72 8.57 ±0.55 0.026* Total egg distance (1–7) 39.67 ±3.57 56.44 ±3.16 0.001** Mean of egg distances 6.61 ±0.59 9.41 ±0.53 0.001** Total number of eggs 23.75 ±1.66 33.55 ±1.77 0.000** Oviposition period 10.15 ±0.18 11.4 ±0.15 0.000**

Table 12. The total number of eggs, egg distances (mm) and oviposition period (day) of T. urticae females when received odours related to predator (P. persimilis /T. bagdasarjani)-prey interactions in their preconditioning and oviposition containers

P. persimilis presence T. bagdasarjani presence Source of variation P (Mean ± SE) (Mean ± SE) 1–2 5.17 ±0.71 5.75 ±1.05 0.652 2–3 6.05 ±0.61 6.62 ±1.07 0.643 Pairwise egg 3–4 4.85 ±0.83 6.57 ±1.09 0.217 distances 4–5 5.87 ±0.80 6.57 ±1.07 0.604 5–6 4.72 ±0.57 7.67 ±0.89 0.008* 6–7 6.95 ±0.57 6.47 ±0.72 0.608 Total egg distance (1–7) 33.62 ±2.73 39.67 ±3.57 0.187 Mean of egg distances 5.6 ±0.45 6.61 ±0.59 0.187 Total number of eggs 19.2 ±0.71 23.75 ±1.66 0.016 Oviposition period 10.15 ±0.21 10.15 ±0.18 1.000

Discussion

Our experiments showed that when T. urticae females perceived the presence of a predator (either P. persimilis or T. bagdasarjani) directly in their predonditioning container, they reduced their oviposition rate, either when they received the odours related to predator during oviposition or even when no signal related to predator was present in their oviposition container. Our suggestion is that the spider mites here have had direct (predator cues) and indirect (dead conspecifics) means of predation risk recognition. This is in consistent with Grostal and Dicke (1999) who demonstrated that fewer spider mites foraged and laid eggs on predator exposed discs in comparison with the discs with intact conspecifics. Lemoset al. (2010) also demonstrated that although Tetranychus evansi Baker & Pritchard web mediated anti-predator behavior, the prey

77 Mona Moghadasi, Azadeh Zahedi Golpayegani, Alireza Saboori, Hossein Allahyari, Hamideh Dehghani Tafti

did not produce denser webs when received cues related to their predator, Phytoseiulus longipes Evans, but laid more eggs in the web far from the predator foraging area. According to our observations, T. urticae females put significantly fewer eggs when perceived the presence of P. persimilis (in their preconditioning container) in comparison with when T. bagdasarjani (an indigenous species) was present. Our interpretation is that T. urticae females interacted with each of the predator species for 24 hours before their oviposition pattern started to be recorded. During this period, T. bagdasarjani might have been recognized as a less harmful predator or a predator that could not deal with the prey web structure as well as P. persimilis. This probable short time experience (Hackl, Schuasberger, 2014) could lead to a higher oviposition rate of T. urticae in the oviposition container especially when no predator (T. bagdasarjani) or no cues related to predator was present in. This is similar to Lemos et al. (2010) who demonstrated that some predators (namely P. persimilis) could cope with the silken web produced by their prey. As far as we know, no published data is present about the reaction of T. bagdasarjani to T. urticae web structure. In the treatments which T. urticae females not only had experienced the predator (P. persimilis/ T. bagdasarjani) presence in their preconditioning container but received odours related to predator-prey interactions in their oviposition container, they reduced their oviposition rate remarkably. This reduction was more perceptible when P. persimilis was chosen as predator species (approx. 44% in comparison with control). Although again the significant reduction of T. urticae oviposition rate was clear in the presence of T. bagdasarjani (25.55 ±6.2) receiving odours during oviposition did not affect the reductionof oviposition rate (24.55 ±7.6) significantly (t = 0.455, df = 38, P = 0.652). Receiving odours related to predator-prey interactions in the preconditioning container (instead of predator direct presence) led to a significant reduction in T. urticae oviposition rates both when the ovipositing females did not receive any odour in their oviposition container and when they still perceived cues related to predator- prey interactions during oviposition. This is similar to Skaloudova, Zemek and Krivan (2007) who demonstrated that prey reduced its activities (i.e. searching for food) when received the cues related to its predator, so that the physiological changes, happened due to food shortage, reduced its oviposition rate. They also exposed T. urticae to various cues of predator and observed that as much as the risk would increase the oviposition rate would decrease. Choh et al. (2010) also confirmed that spider mites laid less egg in the presence of predators, although the predator cues alone (without the prey presence) could not elicit such a response in spider mites. In our experiments, the significant gap between the oviposition rates in the presence of odours related to P. persimilis-T. urticae interactions and in the presence of odours related to T. bagdasarjani-T. urticae interactions were narrower in comparison with that recorded in the first seriesof experiments (direct effect). It seems that when femaleT. urticae directly interacted with its predator and conspecifics which had affected by predator (first series of experiments), especially P. persimilis, it changed its oviposition rate more strictly. Egg placement and egg aggregation by a mother has mostly investigated in predators and parasitoiids who need to avoid kin cannibalism (Lopez-Sepulcre, Kokko, 2002; Schausberger, 2005). Faraji et al. (2000) showed that females of Iphiseius degenerans produced eggs in clusters (an extreme form of egg aggregation, Schausberger, 2005) and related females deposited eggs closer than the unrelated ones did. Chittenden and Saito (2001) demonstrated that phytoseiid mites with greater number of eggs lay eggs closer to each other. To our knowledge, rare investigations have targeted the egg aggregation in herbivorous mites. Thus our study is of the first providing

78 Tetranychus urticae changes its oviposition pattern in the presence of the predatory mites... evidence that females of T. urticae tend to lay eggs in significantly closer distances from each other when they realize the predator (either P. persimilis or T. bagdasarjani) presence. Interpreting the significant reduced pairwise distances between eggs mostly recorded from the eggs 1–4 and not-significant distances mostly recorded from the latest ones, we need to design new experiments. Our results showed that when T. urticae females received odours related to predator-prey interactions in their oviposition patch, regardless of being affected by predator directly or indirectly (in their preconditioning container), they decreased the pairwise egg distances in at least 4 and at most 6 pairs of eggs, while when odours were absent in the oviposition container, the distances decreased in at least 2 and at most 3 pairs of eggs. Thus it seems important for the ovipositing T. urticae to perceive risk at the time of (in coincident with) laying eggs. When T. urticae females lay their eggs closer to each other, they reduce their movement activity during oviposition and consequently the female exposure to the predator and the risk of predation would decrease. Little is known about whether the time of risk experience would affect the spider mite oviposition pattern. Zeraatkar, Zahedi Golpayegani and Saboori (2013) discussed the effect of relatedness on egg distances in three samples of P. persimilis. They found that when two female predators were introduced to the same patch, the distance of second female eggs from the first ones was not affected by kinship. Chittenden and Saito (2001) reported about the variation in egg-laying patterns between the species of Phytoseiidae and correlated the feeding type and egg distances but, rare documents have reported the variation of oviposition pattern in spider mites or even any herbivorous mite species. Several studies have demonstrated the effect of prey mite species on the biological aspects of the predators (Dicke, Sabelis, de Jong, 1988; Gnanvossou, Yaninek, Hanna, Dicke, 2003) but to our knowledge, little information has addressed whether the predator species could alter that in spider mites. Hackl and Schausberger (2014) noted delayed and/or decreased oviposition could be of typical antipredator responses of spider mites. Fernández Ferrari and Schausberger (2013) showed that T. urticae laid fewer eggs in the presence of cues of P. persimilis and Amblyseius andersoni, but they did not investigate the probable changes of prey oviposition period in such situations. Our study showed that the oviposition period of T. urticae was reduced significantly either in direct predator (P. persimilis/ T. bagdasarjani) presence or when females received odours related to predator-prey interactions in their preconditioning container, even if the odours were not present in their second container. Although T. urticae oviposition period was shorter in the treatments with P. persimilis (in comparison with those with T. bagdasarjani), this difference was only significant in the direct presenceof a predator, when the odours related to predator-prey interactions were present in the oviposition container. Reducing the oviposition period along with less oviposition rate (mentioned previously) could be considered as an adopted strategy to decrease the risk of predation. Predation could change the behavior, biology and morphological characters of prey individuals. As a response, prey could develop a wide range of adaptations to reduce the risk of predation. It is known that spider mites are able to detect predators by chemical cues they receive (Grostal, Dicke 1999). Here, we showed that such recognition could induce some types of antipredator behaviors, i.e. oviposition rate reduction, manipulating the distances between eggs and reducing the oviposition period. Our results are of rarely published information about the antipredator behaviors of spider mites.

79 Mona Moghadasi, Azadeh Zahedi Golpayegani, Alireza Saboori, Hossein Allahyari, Hamideh Dehghani Tafti

Acknowledgement We are grateful to Masoud Rezaei Adariany for his valuable help in rose cultivation. The pro- ject was partly supported by a grant from “Center of Excellence of Biological Control of Pests”, Department of Plant Protection, Faculty of Agriculture, University of Tehran, Karaj, Iran, which is greatly appreciated.

References

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Cite as: Moghadasi, M., Zahedi Golpayegani, A., Saboori, A., Allahyari, H., Dehghani Tafti, H. (2019). Tetranychus urticae changes its oviposition pattern in the presence of the predatory mites, Phytoseiulus persimilis and Typhlodromus bagdasarjani. Acta Biologica, 26, 65–81. DOI: 10.18276/ab.2019.26-07. #1#

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Acta Biologica 26/2019 | www.wnus.edu.pl/ab | DOI: 10.18276/ab.2019.26-08 | strony 83–97

Characteristics of a new variant of rabbit haemorrhagic disease virus – RHDV2

Dominika Bębnowska,1 Paulina Niedźwiedzka-Rystwej2

1 Student’s Scientific Circle of Immunology; Institute of Biology, University of Szczecin, Felczaka 3c, 71-412 Szczecin, Poland 2 Institute of Biology, University of Szczecin, Felczaka 3c, 71-412 Szczecin, Poland

Corresponding author, e-mail: [email protected]

Keywords rabbit haemorrhagic diesease virus, RHDV2, GI. 2 Abstact RHD (Rabbit Haemorrhagic Disease) is an etiologic agent that causes viral haemorrhagic disease of rabbits, which is also referred to as rabbit plague. The desire to explore knowledge about this pathogen in the world results not only from its similarity to human hemorrhagic fevers, for which RHDV can be considered as a good research model. It is also important that with myxomatosis, rabbit plague is the most severe viral disease of these animals. Describing the classic RHDV virus, followed by RHDVa, which is considered the first described antigenic variant of the RHD virus, allowed to become familiar with the information that until now constituted the basic building block of the control of viral haemorrhagic disease of rabbits in the world. However, the attributes of RHDV2, characterized only in 2010, differ significantly from the specifics of RHDV strains known to date. What’s more, the global expansion of this pathogen has significantly changed the RHD image, the methods of its identification, control and eradication. The completely changed face of the virus that causes rabbit plague causes that the belief about full supervision over RHD is now obsolete.

Charakterystyka nowego wariantu wirusa choroby krwotocznej królików – RHDV2 Słowa kluczowe wirus choroby krwotocznej królików, RHDV2, GI. 2 Streszczenie Wirus RHD (Rabbit Haemorrhagic Disease) stanowi czynnik etiologiczny wywołujący wirusową chorobę krwotoczną królików, którą określa się również jako pomór. Chęć do zgłębiania wiedzy na temat tego patogenu na świecie wynika nie tylko z jego podobieństwa do ludzkich gorączek krwotocznych, dla których RHDV może być uznany za dobry model badawczy. Istotny jest także fakt, że wraz z myksomatozą, pomór królików to najdotkliwsza choroba wirusowa tych zwierząt. Opisanie klasycznego wirusa RHDV, a następnie RHDVa, który uznawany jest za pierwszy opisany wariant antygenowy wirusa RHD, pozwoliło na zaznajomienie się z informacjami, które dotychczas stanowiły podstawowy budulec bastionu dla kontroli nad wirusową chorobą krwotoczną królików na świecie. Jednakże cechy scharak- teryzowanego dopiero w 2010 roku RHDV2 znacznie odróżniają się od specyfiki znanych do tej pory szczepów RHDV. Co więcej, globalna ekspansja tego patogenu znacząco zmieniła

83 Dominika Bębnowska, Paulina Niedźwiedzka-Rystwej

obraz RHD, metody jej identyfikacji, kontroli i zwalczania. Całkowicie zrewoltowane oblicze wirusa wywołującego pomór królików powoduje, że przekonanie o pełnym dozorze nad RHD jest już przestarzałe.

Introduction

RHD (Rabbit Haemorrhagic Disease) is an etiologic agent that causes viral haemorrhagic disease of rabbits, also called plague. Rabbits, in addition to being a key element of the economy of many countries, also exist as an important link in the trophic chains of the Mediterranean ecosystems. As laboratory animals, they are widely used in various types of research and diagnostic analyzes (Abrantes, van der Loo, Le Pendu, Esteves, 2012). The huge losses in their numbers, which started the first documented outbreak of the RHD epidemic in China in 1984 (Liu, Xue, Pu, Quian, 1984), led to serious economic losses as well as decline in the number of endangered species for which rabbits form the basis of food. This situation led, among others, to the fact that RHDV has become the object of research around the world (Abrantes et al., 2012). It was observed that the experimental infection of rabbits with this pathogen is a research model in the pursuit of knowledge about human hemorrhagic fevers. The key fact that allows this factor to be used is that the rabbit hemorrhagic diseases virus infections in rabbit and human are characterized by similar clinical features, including the equally rapid course of infection. These concordants mean that research into rabbit fever virus provides a perspective for the development of new effective methods of fight and prevention in the future of human hemorrhagic fevers (Tokarz-Deptula, Deptula, Kesy, 2002). In addition, pathomorphological changes in the liver, observed post-surgically among the experimentally infected RHDV rabbits, are the subject of research in the failure of this organ, which refers to the pathogenesis of viral hepatitis observed in humans, as well as in determining the effectiveness of hepatoprotective agents (Pazdzior, Otrocka-Domagala, Rotkiewicz, Drzewiecka, 2011). In addition, RHDV is a virus whose genetic material is a single strand of RNA with positive polarity, and RNA viruses are characterized by high genetic variability caused by frequent muta- tions within the strand encoding genetic information (Fitzner, Niedbalski, 2017). The consequence of such phenomena is diversity among isolated strains of viruses, which is observed in the case of viruses that cause haemorrhagic disease of rabbits. Thanks to this, it is also possible to consider the factors that cause RHD as a tool for research conducted to expand the current state of knowledge about RNA viruses, among which there are many pathogens of humans and other mammals. The purpose of the work is to characterize a relatively recently registered RHDV2 virus, the features of which differ significantly from the specifics of the previously known RHDV strains. The economic, epidemiological and diagnostic situation will be evaluated, which will allow conclusions to be drawn about the threats that may be caused by the emergence and expansion of the RHDV2 in the world.

History of appearance in RHDV2

Since the end of 2010, deaths have been reported among vaccinated and non-vaccinated rabbits in Northwest and Northern France (Le Gall-Recule et al., 2011). Sectional studies of dead animals reveal various changes in many organs, characteristic of infections that trigger RHDV. The factor responsible for these incidents was subjected to many analyzes and that allowed for demonstration its kinship with the RHDV and RHDVa virus (Le Gall-Recule et al., 2011).

84 Characteristics of a new variant of rabbit haemorrhagic disease virus – RHDV2

However, never previously observed properties in any RHDV strain (neither RHDV nor RHDVa), led to the conclusion that it belongs to a different gene group and exists as a virus that causes haemorrhagic disease of rabbits (Le Gall-Recule et al., 2011). This factor was also diagnosed by another research team in Spain (Dalton, Nicieza, Abrantes, Esteves, Parra, 2014). In 2011, in this country, people started to observe the emergence of powerful outbreaks of RHD infection, adults as well as juveniles. The new strain, called RHDV-N11, was quickly transferred to many Spanish provinces, replacing the predominant G1 RHDV genotypes. His presence led to huge losses in the rabbit industry, putting him in a serious economic disadvantage (Dalton et al., 2014). The results of the analyzes carried out, showed a significant phylogenetic distanceof strain led to introduce the new term for RHDV-N11, namely RHDVb, which is assigned to the RHDVb group (Dalton et al., 2012). Next, reports of a new strain have appeared in the summer of 2011 in Italy. The incident occurred in the north-eastern part of this country in one of the industrial farms (Le Gall-Recule et al., 2013). At the beginning of the next year, outbreaks of disease began to be observed also among wild populations (Velarde et al., 2017). Based on facts regarding the unique characteristics of these strains, the pathogen was called RHDV2 (Le Gall-Recule et al., 2013). The extent of this lagovirus throughout the Iberian Peninsula confirms its appearance in November 2012 also in Portugal, where he was responsible for epidemics among wild rabbit populations (Abrantes et al., 2013). The presence of the virus again posed a threat to the endemic species inhabiting this area, i.e. Iberian lynx and the Iberian imperial eagle (Carvalho et al., 2017; Silverio et al., 2018). RHDV2 have covered a relatively large part of the continent quite quickly. In May 2013, he was diagnosed, among others in Sweden, in samples taken during fieldwork (Neimanis et al., 2018). List of countries reporting cases of infections issued in Great Britain (Westcott et al., 2014). and Scotland in 2014 (Baily, Dagleish, Graham, Maley, Rocchi, 2014), Germany (information FLI- Friedrich Loeffler Institut, 2013), as well as reports submitted to the World Organization for Animal Health, Norway (OIE WAHIS, 2014), Switzerland (OIE WAHID, 2016a), Finland (OIE WAHIS, 2016b) and Denmark (OIE WAHIS, 2016c) (Velarde et al. 2017). In Tenerife, virus has been observed since 2015 (Martin-Alonso, Martin-Carrillo, Garcia-Livia, Valladares, Foronda, 2016). The detection of the RHDV2 virus in Poland is associated with the study of rabbits that died in September 2016 on a farm in the village of Reduchów near Zduńska Wola and individuals who died in June 2017 in the West Pomeranian Voivodeship (Fitzner, Niedbalski, 2018). It was also found in studies conducted in slow-breeding rabbit populations and individuals from farms, that this factor is characterized by a tendency to displace the classical RHD variant both in Tenerife and also in the Iberian Peninsula (Le Pendu et al., 2017; Silverio et al., 2018; Velarde et al., 2017), as well as in France and the Azores (Duarte et al., 2015; Martin-Alonso et al., 2016). The Azores are also places where RHDV2 was first diagnosed outside the European continent in 2014 (Duarte et al., 2015). The virus appeared in Australia in Canberra in May 2015 (Hall et al., 2015), where it also appeared in the southern part of the country, when it was detected on December 1, 2015 (Peacock et al., 2017). The presence of the pathogen has led to a reduction in the number of rabbits by about 80% in this area, which in fact can have a positive impact on the environment here, due to the recognition of these animals as pests (Mutze et al. 2018). In 2016, the presence of RHDV2 was also confirmed in Oceania and Canada (Rouco, Aguayo-Adan, Santoro, Abrantes, Delibes-Mateos, 2019). The RHDV2 virus was also detected in Africa, where it was confirmed in 2015 in Benin, Tunisia (Rahali, Sghaier, Kbaier, Zanati, Bahloul, 2019), and also in 2017 in Morocco (Lopes, Rouco, Esteves, Abrantes, 2019). Since 2017, RHDV2 has been also reported in New Zealand and the United States. In 2018, the virus appeared in Israel (Rouco et al., 2019).

85 Dominika Bębnowska, Paulina Niedźwiedzka-Rystwej

RHDV2 – a new member of the genus Lagovirus

Phylogenetic analyzes as a basic tool for determining the relationship of the tested materials are an important point in the process of identifying viral genetic material isolated posthumously from infected rabbits, as evidenced by research carried out by many scientific teams (Abrantes et al. 2013; Camarda et al., 2014; Dalton et al., 2015, Dalton et al., 2018; Dzialo, Deptula, 2015; Fitzner, Niedbalski, 2017; Le Gall-Recule et al., 2003, 2011; Lopes et al., 2015, 2018). The latest nomenclature aimed at organizing the naming of RHDV, RHDVa and RHDV2/b, based on phylogenetic compounds within the Lagovirus genus, has classified RHDV2 as a separate genotype called GI.2 (Le Pendu et al., 2017). However, the research already carried out at the beginning of the appearance of the putative variant revealed that it is genetically separated from the classical RHDV and RHDVa, because the homology between them is only 87.5% (Le Gall-Recule et al., 2011). It remains inso facto closer associated with non-pathogenic rabbit calicivirus RCV (Dalton et al.,2012; Dzialo, Deptula, 2017). Based on the studies of the structural gene sequence encoding the VP60 and VP10 proteins, it was concluded that it constitutes a separate genetic group (Le Gall- Recule et al. 2013), located between apatogenic strains RCV-A1 and weakly pathogenic MRCV, which confirms close relation to RCV (Fitzner, Niedbalski, 2017). In addition, the classification of the variant as a separate member of the Lagovirus family confirms its distinctive from classical RHDV and RHDVa structure, encompassing amino acid differences in the P domainof the capsid protein (Leuthold, Dalton, Hansman, 2015) affecting the specificityof HBGA- (Histo-Blood Group Antigen) binding, which may be associated with its antigenicity (Le Gall-Recule et al., 2013). Antigenic characterization is another aspect that distinguishes RHDV from each other and has an impact on the classificationof variant 2 as a separate position within the species (Le Gall-Recule et al., 2013). These discrepancies have been demonstrated, inter alia, in studies via monoclonal antibodies (mAbs) in the Dot Blot technique, as well as hemagglutination reactions (Dalton et al., 2012). The antigenic differences exhibited by RHDV2 and RHDV were also confirmed by the generation and evaluation of RHDV2 specific VLP particles (Barcenaet al., 2015). The fact that only a partial cross-protection within the variants of the virus has been disclosed. It is noted that the observed lethal course of infection among individuals who had previously had RHD disease, as well as the lack of efficacy of vaccines used against RHDV, confirms the individual serotype of the new pathogen (Dalton et al., 2012; Le Gall-Recule et al., 2013). Also significant is the variability in the course of RHDV2-induced disease, mainly concerning the duration of the disease, mortality, as well as the increased frequency of sub-acute/chronic forms of the disease (Fitzner, Niedbalski, 2017; Le Gall-Recule et al., 2013). It is worth noting that the source of the appearance of RHDV2 is not fully understood, because there are many hypotheses trying to solve this dilemma, among others the hypothesis of evolution from another lagovirus, as well as the genetic jump hypothesis. However, some molecular data cross the assumption that RHDV2 is the result of genetic evolution of a known representative of the Lagovirus family, which causes that genesis of the variant is still questioned (Le Gall-Recule et al., 2013).

Characteristics of RHDV2

RHDV2 virus molecule is not different compared to the previously described classical variant of the RHD virus. The new virus genome is analogously contained in two open reading frames (ORFs), where ORF1 encodes unstructured genes and participates in the synthesis of the VP60 protein, while ORF2 encrypts the genes responsible for the structural VP10 protein (Mahar et al.,

86 Characteristics of a new variant of rabbit haemorrhagic disease virus – RHDV2

2017). The average amino acid homology between viruses, which is 89.2%, drops to 60% the comparison is limited to the 7 regions of the P domain, that show the highest variability. Thanks to analyzes in crystallographic resolution of P domain of VP60 protein, it was possible to show structural differences between pathogens These discrepancies, in spite of the relatively similar design of the helix, relate to the deployment of the P1 subdomain of helix, which is more displaced. There is also a different conformation in the P2 domain, on the surfaceof which on the elongated loops there are amino acid changes conditioning the differences between RHDV2 and RHDV (Barcena et al., 2015).

Epidemiology of RHDV2

Natural and experimental RHDV infections observed in the past always concerned only one characteristic host – the European rabbit Oryctolagus cuniculus. The emergence and spread of RHDV2 strains in the environment revealed that it has the ability to infect a much wider range of species, not limited to O. cuniculus, which is a real phenomenon within lagovirus. This factor was diagnosed as the cause of acute epidemics among different Leporidae. In Sardinia, cases of disease similar in terms of symptoms to EBHS have been reported, among individuals belonging to Lepus capensis (Puggioni et al., 2013). Laboratory analyzes using a direct haemag- glutination test, using human blood cells, an ELISA test, as well as sequencing of the VP60 protein genetic code, absolutely ruled that the causative agent of these epizootics is RHDV2 (Puggioni et al., 2013). Isolated cases of disease caused by the new lagovirus have also been described among Lepus corsicanus (Camarda et al., 2014). A similar situation was observed for European brown hares Lepus europaeus on the European continent (Hall et al., 2018; Velarde et al., 2017), as well as in Australia (Hall et al., 2017). Despite pathological changes consistent with EBHSV, described during performed autopsies of dead animals, examination of isolated genetic material by means of RT-PCR and tests based on specific monoclonal antibodies-mAb, showed the presence of RHDV2 virus in samples (Velarde et al., 2017). The analysis of data on the impact of the virus on different lagomorphs populations revealed that this pathogen is highly pathogenic to European rabbit (O. cuniculus), European hare (L. europaeus) and to L. capensis, while lower pathogenicity is characterized by towards L. corsicanus. There were also cases of infection of L. timidus (Neimanis et al., 2018) however, the level of virulence of RHDV2 strains against this species has not been determined (Le Gall-Recule et al., 2017). It is also worth mentioning that this virus causes mortality at a similar level, both for young and adults. The sex of animals is irrelevant to the virulence of the virus, which is analogous to the situation observed with infection caused by classical RHDV (Rouco et al., 2018). Transmission of the RHDV2 virus can be described as typical, which is observed in the case of R H DV. The presence of RHDV2 has been detected also in the carcase of the Mediterranean Pine Vole (Microtus duodecimcostatus) and the White-Toothed Shrew (Crocidura russula). An experimental infection of rabbits with an inoculum obtained from said animals showed that these animals are capable of passive transmission of the virus. This finding is also confirmed by the belief that RHDV2 breaks the interspecies barrier (Calvete et al., 2019).

87 Dominika Bębnowska, Paulina Niedźwiedzka-Rystwej

Immunological processes occurring during RHDV2 infection

Studies have been carried out in which a significant participationof CD4+ and CD8+ T lymphocytes have been demonstrated in the mechanisms of specific cellular response of rabbits exposed to RHDV2 infection. This fact has also been confirmed by the increased numberof these cells in a short time after the use of protective vaccination, which further induces the production of specific antibodies as humoral response mechanisms. In addition, early activation of B and T lymphocytes as well as macrophages was observed in animals that did not show any pathological symptoms after contact with the pathogen, which in turn leads to enhanced proinflammatory cytokine activity (Muller, Ulrich, Schinkothe, Muller, Kollner, 2019).

Clinical symptoms and course of infection caused by RHDV2

Despite some differences regarding the clinical features of RHDV2 infection as compared to RHDV, the symptoms of new pathogen infection remain consistent with the classic variant (Duarte et al., 2015). Macroscopic observations and performed autopsies showed the presence of typical changes in many organs of various species hosts. Nose bleeding and congestion of the tracheal mucosa and lungs are recorded. In addition, a foamy-bloody discharge is present in the trachea. Irregularly located pulmonary embolism, bloody ecchymosis and moderate to severe intracapillary hemorrhage is observed. The pathology of the liver refers to multifocal changes with the presence of irregular lesions in the entire parenchyma, which manifests itself in characteristic marbling discolorations. The organ is enlarged, pale and fragile, which is characteristic of RHD. Microscopic examination also reveals fat degeneration of hepatocytes and multinucleation of these cells (Abade Dos Santos et al., 2017; Capucci, Cavadini, Schiavitto, Lombardi, Lavazza, 2017; Carvalho et al., 2017; Duarte et al., 2015; Duarte et al., 2015;Lopes et al. 2014; Martin-Alonso et al., 2016;Puggioni et al., 2013;Simpson, Everest, Westcott, 2014;Velarde et al., 2017). The spleen is dark and swollen, and necrotic changes affect the red and white pulp (Puggioni et al., 2013; Velarde et al., 2017). In the kidneys, the modifications concern the epithelial liningof the proximal tubules of the nephron, which is described as necrotic (Carvalho et al., 2017; Carvalho et al., 2017; Martin-Alonso et al., 2016; Simpson et al., 2014, Velarde et al., 2017). Areas of diminished tissue and inflammation are also found in the intestinal villi of the small intestine (Abade Dos Santos et al., 2017; Duarte et al., 2015; Puggioni et al., 2013). In addition, there are haemorrhages and congestions in the heart as well as in the thymus (Duarte et al., 2015; Lopes et al. 2014). Moreover, in all animals, similar to disease caused by RHDV and RHDVa, the phenomenon of disseminated intravascular coagulation – DIC (Carvalho et al., 2017; Duarte et al., 2015) is recorded in small capillaries and pulmonary arteries. Laboratory blood tests of two rabbits with RHDV2 infection were performed, followed by the analysis of biochemical parameters, significant changes were found within them. Unlike in the course of infection induced by classical RHD virus, low values of hepatic enzymes, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT), have been found in blood serum, which may result from reduced production of these enzymes by damaged hepatocytes, or it may be effect of the short half-life of these parameters. In the case of the first rabbit there was a slight decrease in cholesterol level, hypoalbuminemia, as well as high hypoglycemia. Analysis of blood belonging to the second rabbit showed hyperglyceridemia and hypercholesterolemia in his case. Both animals showed elevated levels of gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP), bilirubin and bile acids. Analysis of the electrolyte level in the case of the

88 Characteristics of a new variant of rabbit haemorrhagic disease virus – RHDV2 first rabbit showed moderate hypochloremia and hypokalemia. In the study, in contrast to the values observed in the case of RHDV infection, a significant increase in fibrinogen levels was demonstrated for both rabbits. It was determined that the prothrombin time (PT) and activated partial thromboplastin time (APPT) is increased, which in turn remains consistent with the results observed with the classical RHD virus (Bonvehi et al., 2019). In the course of RHDV2 infection, as in the case of RHDV-induced infection, a reduced amount of leukocytes is observed in the liver, which together with the fact that RHD rapidly el- evates the temperature above 39°C, indicates the development of systemic inflammatory response syndrome – SIRS. It should also be mentioned that the observed phenomenon of impaired immune response is associated with necrotic changes in the spleen, progressive apoptosis and the initial increase in CD4 + and CD8 + T cells followed by a significant decrease in the number of these cells. It has been demonstrated that within 36 hours of experimental animal infection the level of total T-cells in blood decreased to approximately 50% for CD4 + T cells and to approximately 10% of normal values for CD8 + T cells (Muller et al., 2019). Infections caused by the RHDV2 virus, despite the similarity in the observed disease symptoms and caused by anatomopathological changes, are significantly different from those caused by classical RHDV. These discrepancies relate mainly to the duration of the disease, mortality observed, as well as the incidence of subacute and chronic forms of the disease. Experimental inoculation of laboratory animals revealed that symptoms of hemorrhagic fever started appearing 3 to 9 days after inoculation and lasted on average 5 days, which is a much longer period compared to the typical course of RHD. A decreased frequency of acute course of the disease was also noted, with an increase in the frequency of subacute and chronic forms, which were characterized on the basis of morphological symptoms (Westcott et al., 2014). It was also observed that this virus is responsible for the development of the disease not only in adult rabbits, about 40–50 days old, but also in animals under 30 days. The high virulence of RHDV2 strains confirms the finding of the development of disease, initiated under experimental conditions, in individuals aged 11 days (Dalton et al., 2014). Studies have been carried out that showed that RHDV2 is able to suppress the innate immune mechanisms associated with infection in juveniles, thanks to which progression of infection is possible (Neave et al., 2018). It was generally accepted that RHDV2 is less virulent for adult animals, since the mortality rate oscillates between 0–75% (Rouco et al., 2018), where for classical RHDV it is above 90%. The generalized value of the mortality rate for a new virus, after considering all age classes, varies between 20–50% (Dalton et al., 2012). However, it should be noted that some strains of RHDV2 have been isolated, inducing mortality at about 80%, which is similar to the typical course of RHD. This provides the supposition that recombination events in the course of virus evolution have led to the emergence of high pathogenicity strains that spread in the environment (Capucci et al., 2017) and are promoted by selective pressure (Neimanis, Larsson Pettersson, Huang, Gavier-Widen., Strive, 2018). It should be mentioned that the analysis of the condition of animals affected by rabbit haemorrhagic disease, caused by the RHDV2 virus, unambiguously determines that death of individuals in the course of the plague is a consequence of progressive multi-organ failure and described during the RHDV infecion-DIC (Capucci et al., 2017; Carvalho et al., 2017).

Diagnostics and immunization

The emergence and prevalence of RHDV2 has led to the compulsion of developing new, more sensitive diagnostic methods to identify this pathogen. The fact that the symptoms of the disease

89 Dominika Bębnowska, Paulina Niedźwiedzka-Rystwej caused by this virus are identical to the symptoms caused by RHDV, makes the diagnosis that differentiates these factors via the pathological changes is impossible (Duarte et al., 2015, McGowan, Choudhury, 2016). It is also important that due to the high specificity of tests used against the classical RHD virus, in the confrontation with RHDV2, the results remain negative (Mahar et al., 2017). The problem in the selection of appropriate vaccines, during emerging RHD epidemics, has its solution in the application of additional diagnostic methods, allowing to determine the type of pathogen. This is mainly aimed at reducing the costs incurred, as well as increasing the effectiveness and speedof struggle in suppressing still emerging disease foci (Dalton et al., 2018). The most effective option seems to be molecular techniques related to the genome of viruses (Duarte et al., 2015). They can also be used to detect recombination events, which are an important tool for enriching knowledge about the virus (Dalton et al., 2018). Unfortunately, awareness of the effectivenessof the vaccine obtained from the liver of infected rabbits remains limited and, due to the high sensitivity of the nucleic acid, requires additional research methods (Carvalho et al., 2017). It was observed that false positive results were obtained in the polymerase chain reaction of PCR with reverse transcriptase (RT-PCR), which was provoked by genetic material of inactivated vaccine (Carvalho et al., 2017). It has now been confirmed that the described new RT-PCR tests are an effective tool for controlling the spread of the virus (Dalton et al., 2018). There were also effective modifications of this method for recognizing lagoviruses. They are successfully used in, among others Australia, where the presence of four lagoviruses is observed, i.e. GI.1c, GI.2, GI.1a-Aus and GI.1a-K5 (Le Pendu et al., 2017). These methods include multiplex RT-PCR as well as RT-qPCR in real time (Quantitative reverse transcriptase real-time PCR) (Hall et al., 2018). It should be noted that the RT-qPCR method stands out against its others with its particularly high sensitivity, as it detects only ten particles of viral RNA, and the whole process lasts under 3 hours (Duarte et al., 2015). The huge disadvantage of this type of analysis is the necessity of specialized equipment, which results in a lack of chance to diagnose the pathogen directly in the place of its presumed presence. The immunochromatographic test (LFA) has been developed as an option enabling such activities. Identification of the causative agent with LFA takes about 10 minutes, however, it is less sensitive than the RT-PCR method (Dalton et al., 2018). A big drawback of this system is the inability to analyze multiple samples simultaneously, as well as quantifying the antigen, which certainly prevents reliable control of the evolution of the disease. Another diagnostic method is the enzyme-linked immunosorbent assay (ELISA), based on the use of the mAb 2D9, which is characteristic for RHDV2. This method is characterized by 100% sensitivity and over 97% specificity for the RHDV2 antigen, and its use positively affects the reduction of research costs and the functioning of diagnostic laboratories (Dalton et al., 2018). Unfortunately, for the efficient and effective control of the virus, there is a need for continuous improvement of diagnostic methods. The current lack of high quality rapid tests on the market, intended for use in private breeding is an extremely unfavorable situation. The time elapsing from the detection of a disease outbreak, until the results are obtained and the implementation of the procedure limiting the spread of the pathogen, is prolonged, which significantly reduces the dynamics of control over emerging RHD epizootics. Difficulties that have arisen with the spread of infections caused by RHDV2 strains are increasing as knowledge about this pathogen increases. The observed lack of protection against the development of the disease, among individuals vaccinated against RHDV, proved the exist- ence of immunogenic differences between viruses.In addition, the mortality observed in rabbits diagnosed with RHD that have had infection with classical strains of the virus and acquired immunity against RHDV in this way, shows the lack of cross-protection between RHDV2 and

90 Characteristics of a new variant of rabbit haemorrhagic disease virus – RHDV2

RHDV. This fact was of great importance for the new pathogen, which obtained such a large range in a relatively short time (Le Gall-Recule et al., 2013; Peacock et al., 2017), which is why it became urgent to develop a vaccine against RHDV2. The inactivated vaccines introduced at the beginning were prepared from rabbit liver extracts that were infected under experimental conditions. Their efficacy was assessed through studies conducted on 4-week-old female rabbits using four different strains of the RHDV2 virus (Pacho, Dahdouh, Merino, Suarez, 2016). Vaccines have been recognized in the European Union as a temporary means of combating the pathogen and controlling the epidemic. However, due to the low mortality rate generated by the virus, there were concerns about the amount of viral antigen obtained and thus the effectiveness of vaccination. As a solution to this difficulty, the useof RHDV2 VLP particles was proposed for the development of recombinant vaccines, which are an effective meansof controlling the spread of the virus in the environment (Barcena et al., 2015). The recombinant RHDV2-VP1 vaccine, based on the VP1-capsid protein and produced using a baculovirus expression system, proved to be effective. It has been shown that the inductionof effective immunological protection lasting for at least 14 months takes place 7 days after the immunization, while the revaccination after 21 days did not affect the prolongation of the protection period (Muller et al., 2019). Another problem in the aspect of preventive vaccination concerns the selection of a suitable vaccine. Although many sites have reported progressive replacement of classical RHDV and RHDVa variant by RHDV2 strains (Duarte et al., 2015; Lopes et al., 2014, 2015, 2017; Mahar et al., 2017; Martin-Alonso et al., 2016;Neimanis et al., 2018), there are regions where the coexistence of these pathogens is still detected. This leads to the emergence of a significant difficulty, which is the need to use two types of measures against all factors at the same time. The solution to this problem is differential diagnostics (Dalton et al., 2018).

The consequences of the RHDV2 epidemic

The RHDV2 research, and hence characterization of its unique features, on the basis of which it was considered a separate virus, prompted researchers to develop a new classification and nomenclature system within RHDV. The RHD virus belongs to the genus Lagovirus within the Caliciviridae family, which is a group of viruses without capsules with the genetic material ss- RNA (+) (Dzialo, Deptula, 2017). These viruses were initially classified within the Picornaviridae family, however, taking into account the fact of their unique structure, replication strategy and physicochemical properties, they were identified as a new family. Lagoviruses form a taxon whose genetic length is 7.4–8.3 kb, and the RNA strand is characterized by a polyadenylated 3 ‘end, covalently closed at the 5’ end and two reading frames – ORF. In addition, compared to other members of Caliciviridae, they are distinguished by high pathogenicity and mortality among their host species (Hall, 2002). Due to the successively enriching diversity of lagoviruses, the need to reform the applied system of naming and classification of RHD virus isolates has emerged (Le Pendu et al., 2017). The analyzes carried out in 2002 allowed to describe six main genogroups among the studied RHDV strains (G1–G6). Genogroups G1–G5 contained classical RHDV, while strains of the first known antigenic variant – RHDVa were included in the G6 genogroup. The division into the above-mentioned groups (with the exception of the G6 genogroup) occurred then based on the time distribution as well as the geographical occurrence of isolated strains (Le Gall-Recule et al., 2003). The latest nomenclature suggests the division of lagoviruses based on phylogenetic analyzes based on a comparison of the full sequence of the VP60 structural protein gene. The genogroup GI and GII were thus isolated. Within the first one, among others,

91 Dominika Bębnowska, Paulina Niedźwiedzka-Rystwej genotypes corresponding to RHDV (GI.1) and strains RHDV2/b (GI.2). In addition, the GI.1 gene group concentrates variants corresponding to the previous division, i.e. GI.1a (G6/RHDVa), GI.1b (G1), GI.1c (G2), GI.1d (G3–G5). It is worth noting that within all strains of the RHD virus one can distinguish those with varying degrees of hemagglutination capacity of human red blood cells, which constitute the predominant group, as well as strains devoid of this characteristic (Xue et al., 2016). In the division there is also a GII gene group describing the European brown hare syndrome virus – EBHSV, which together with RHDV forms a kind of Lagovirus (Le Pendu et al., 2017). The deterioration of the economic situation related to the emergence and spread in the 1980s of the classical virus causing haemorrhagic fever of rabbits, was effectively offset by the implemented preventive measures. However, with the occurrence of the first epizootics caused by the new lagovirus-RHDV2, there is still a growing economic loss in commercial and private rabbit farms, as well as in the rabbit food processing industry. It is also reported in some countries about the inconvenience of tourism, where organized rabbit hunting was a tourist asset, which certainly does not favor national economic statuses (Carvalho et al., 2017). The expansion of rabbit haemorrhagic disease virus in the environment still causes a significant reduction in the population of the European rabbit. Recently, there has been a sharp decline in the density of individuals among rabbits, an important link in Mediterranean ecosystems. This led to a loss of balance in the trophic networks of the Iberian Peninsula. Thanks to surveys conducted in this area, it is known that the only factor that causes RHD is RHDV2. A reduced number of Spanish and Portuguese rabbits caused a simultaneous significant lossof individuals belonging to endemic species in this area for which O. cuniculus forms the basis of food. These changes accumulate within the critically endangered Iberian lynx and the Spanish imperial eagle (Dalton et al., 2018; Rouco et al., 2018). It was determined that the decline in the number of rabbits there is 60–70%, in the case of Iberian lynx 65.5%, while the population of the Spanish eagle decreased by 45.5%. These values confirm that the circulating virus has a negative impact on attempts to rebuild the Iberian lynx population in many areas (Monterroso et al., 2016). The presence of RHDV2 strains, due to the increased host panel, may also pose a threat to endangered species of different lagomorphs. This issue should become a subject of interest for researchers in order to prevent the negative impact of the virus on the natural environment (Velarde et al., 2017). The RHDV2 virus from the unexpected appearance in France in 2010 significantly affected the ordered erudition in the rabbit’s haemorrhagic disease. A very important issue is also the wider spectrum of hosts infected with RHDV2. The discussed course of infection caused by this factor, as well as the anatomic changes observed during the disease, indicate that RHDV2 is very difficult to diagnose. This also underlines the reduced mortality in adult rabbits and the prolonged duration of the disease. Difficulties also affect the diagnostic methods developed to quickly and efficiently determine the RHD drive factor. This issue was in fact a priority due to the ineffectivenessof the vaccines previously used against RHDV, which proved ineffective against the RHDV2 virus. This necessitated the development of new specific preventive measures.In addition, the problem is exacerbated by the fact that infections caused by all types of viruses are manifested by the presence of identical symptoms, which excludes the use of disease symptoms as a criterion for strain differentiation. The appearance of a virus has many consequences. One of them is the new system of classification and nomenclature introduced by scientists within the RHD virus. The economic and environmental effects caused by the spreadof RHDV2 in the environment are also frightening. There are negative consequences of its presence in Mediterranean ecosystems, as well as in the economic and economic sphere in countries where the rabbit is a key element of the economy.

92 Characteristics of a new variant of rabbit haemorrhagic disease virus – RHDV2

The appearance of RHDV2 proves that one should not lose vigilance, but still closely monitor the state of the environment. The noticeable tendency for sudden emergence of new disease factors that cause RHD, which causes more and more difficulties in combating them, means that in the future we should expect another opponent who may again disturb the reconstructed harmony.

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95 Dominika Bębnowska, Paulina Niedźwiedzka-Rystwej

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Cite as: Bębnowska, D., Niedźwiedzka-Rystwej, P. (2019). Characteristics of a new variant of rabbit haemorrhagic disease virus – RHDV2. Acta Biologica, 26, 83–97. DOI: 10.18276/ab.2019.26-08. #1#

#0#

Acta Biologica 26/2019 | www.wnus.edu.pl/ab | DOI: 10.18276/ab.2019.26-09 | strony 99–115

A faunistic and ecological characterization of the water mites (Acari: Hydrachnidia) of the Branew River (central-eastern Poland)

Robert Stryjecki

Department of Zoology and Animal Ecology, University of Life Science in Lublin, Akademicka 13, 20-950 Lublin, Poland

Corresponding author, e-mail: [email protected]

Keywords lotic zone, lentic zone, Lebertia rivulorum, synecological groups, species diversity, anthropogenic transformation Abstract A characteristic feature of the Hydrachnidia communities of the Branew River, distinguishing its fauna from that of other Polish rivers, was the very high abundance of species of the genus Lebertia. Species of this genus accounted for as much as 53.1% of the collected material. The most numerous was Lebertia rivulorum, which was caught in numbers not found in other Polish rivers. Lebertia inaequalis and L. oblonga were also abundant. The largest synecological groups were rheophiles and rheobionts (94.9% combined). More individuals (1494) and species (27) were caught in the lentic zone of the river than in the lotic zone (1291 ind., 25 sp.). The species most associated with the lotic zone was L. rivulorum. This species was caught mainly on a substrate of gravel and stones with a small amount of sandy sediments, sparsely covered with Elodea canadensis. The species most associated with the lentic zone was Forelia variegator. The most abundant species in the Branew River, Hygrobates setosus, was caught in much higher numbers in the littoral zone than in the central part of the river. The high similarity of fauna was observed between the lotic and lentic zones, and much lower similarity between sites. The results indicate intensive species migration in the transverse profile of the river and low migration in the longitudinal profile. Higher species diversity was recorded in the lentic zone than in the lotic zone of the river – both in the river as a whole and at the individual sites. In the anthropogenically transformed stretch of the river (straightened riverbed, concrete dam, and concrete bottom), species diversity was significantly lower (H’ = 1.83) than in the natural stretch (H’ = 2.37). The results confirm literature data describing the negative impact of such transformations on Hydrachnidia communities. Despite anthropogenic transformations in parts of the river, the structure of the fauna (a very large proportion of rheobionts and rheophiles), as well as the physicochemical parameters of the water, is indicative of the good ecological condition of the river.

99 Robert Stryjecki

Faunistyczna i ekologiczna charakterystyka wodopójek (Acari: Hydrachnidia) rzeki Branew (środkowo-wschodnia Polska) Słowa kluczowe strefa lotyczna, strefa lenityczna, Lebertia rivulorum, grupy synekologiczne, species diversity, przekształcenia antropogeniczne Streszczenie Cechą specyficzną zgrupowań Hydrachnidia rzeki Branew, odróżniającą jej faunę od innych rzek Polski, była bardzo duża liczebność gatunków z rodzaju Lebertia. Gatunki z tego rodzaju stanowiły aż 53.1% zebranego materiału. Najliczniejsza była Lebertia rivulorum, którą łowiono w liczebnościach niespotykanych w innych rzekach Polski. Ponadto, w dużych liczebnościach łowiono L. inaequalis i L. oblonga. Najliczniejszymi grupami synekologicznymi były reofile i reobionty (łącznie 94.9%). Więcej osobników (1494) i gatunków (27) złowiono w strefie lenitycznej rzeki niż w strefie lotycznej (1291 osobn., 25 gat.). Gatunkiem najbardziej zwią- zanym ze strefą lotyczną była L. rivulorum. Lebertia rivulorum łowiono przede wszystkim na dnie żwirowo-kamienistym z niewielkim udziałem osadów piaszczystych, skąpo poro- śniętym Elodea canadensis. Gatunkiem najbardziej związanym ze strefą lenityczną była Forelia variegator. Najliczniejszy gatunek w rzece Branew – Hygrobates setosus, łowiono zdecydowanie liczniej w strefie przybrzeżnej, niż w centralnej części rzeki. Stwierdzono duże podobieństwa faun między strefą lotyczną i lenityczną i znacznie mniejsze podobieństwa fau- nistyczne między stanowiskami. Uzyskane wyniki świadczą o intensywnej migracji gatunków w profilu poprzecznym rzeki i słabej migracji w profilu podłużnym. Większą różnorodność gatunkową notowano w strefie lenitycznej, niż lotycznej rzeki – zarówno w skali całej rzeki, jak i na poszczególnych stanowiskach. Na przekształconym antropogenicznie odcinku rzeki (wyprostowane koryto, betonowa zapora, wybetonowane dno) stwierdzono znacznie niższą różnorodność gatunkową (H’ = 1.83) niż na odcinku naturalnym (H’ = 2.37). Uzyskane wyniki potwierdzają dane literaturowe opisujące negatywny wpływ takich przekształceń na zgru- powania Hydrachnidia. Mimo lokalnych przekształceń antropogenicznych, struktura fauny (bardzo duży udział ilościowy reobiontów i reofili), a także wartości wskaźników fizyczno- -chemicznych wody, świadczą o dobrym stanie ekologicznym rzeki.

Introduction

A comparison of the number of publications on the Hydrachnidia of standing and flowing water bodies in Poland reveals that lotic ecosystems have been much less researched than lentic ecosystems. Data on the water mites of flowing water bodies in lowland areas of Poland can be found in studies by Pieczyński (1960), Bazan-Strzelecka (1964, 1986), Biesiadka (1970, 1972), Biesiadka and Kasprzak (1977), Kowalik (1981), Cichocka (1996a, 1996b, 2006), Stryjecki (2009, 2010), Stryjecki, Pawlęga, Ścibior (2012), Stryjecki, Bańkowska, Szenejko (2018), Zawal and Sadanowicz (2012), Stryjecki and Kowalczyk-Pecka (2013a), Zawal and Kowalik (2013), Bańkowska, Kłosowska, Stryjecki, Zawal (2015) and Zawal et al. (2017). Characterizations of the Hydrachnidia communities of rivers in upland areas of Poland can be found in works by Kowalik (1981), Kowalik and Biesiadka (1981), Stryjecki and Kowalczyk-Pecka (2013b), Kowalik, Zawal, Buczyńska (2014), and Biesiadka, Kowalik, Ścibior (2015). The least researched by far are the rivers of mountainous areas and foothills. The most important studies on these areas include works by Kupiszewska (1965), Biesiadka (1974, 1979), and Biesiadka and Cichocka (1993). Among the studies cited above, those which take into account longer stretches of rivers or their entire course are of particular value (Bazan-Strzelecka, 1964; Biesiadka, 1970, 1979; Kowalik, 1981; Cichocka, 1996a, 2006; Zawal et al., 2017; Stryjecki et al., 2018). Given that the water mite fauna of flowing water bodies of Poland is less well known than that of standing water bodies, there is a need for research on flowing water bodies to provide a more complete faunistic and ecological characterization of the Hydrachnidia communities of these ecosystems.

100 Water mites (Acari: Hydrachnidia) of the Branew River

In addition to the recognition of the fauna of these ecosystems, the intensification of river research can contribute to a better understanding of the habitat requirements and ecological character of some species, especially taxa found in both flowing and standing waters (e.g.Hygrobates longipalpis (Hermann, 1804), Forelia variegator (Koch, 1837), and Teutonia cometes (Koch, 1837)). Another objective of such research is the provision of new data on the habitat preferences of taxa previously treated as one species, but currently recognized as two (or more) separate species. An example is Hygrobates setosus Besseling, 1942, a sister species of H. nigromaculatus Lebert, 1879. The two species, H. setosus associated with rivers and H. nigromaculatus with lakes, were not ultimately distinguished until relatively recently (Martin, Dabert, Dabert, 2010). Many papers on flowing water bodies (Kowalik, 1981; Kowalik, Biesiadka, 1981; Cichocka, 1996a, 2006; Martin, 1996, 1997; Martin, Speth, 1996; Gerecke, 2002) include H. nigromaculatus in the list of species, although it is highly likely that the data presented pertain to the river species H. setosus. The presentation of further data on the habitat preferences of H. setosus is important because it enables a more complete understanding of the habitat requirements of this species. Research on rivers, especially in areas that have previously been overlooked, can provide new information on species distribution and population sizes. Data from areas that have previously been poorly researched are of particular importance for taxa considered to be rare. Such data contribute to a better understanding of the biology and ecology of these species. Polish rivers have been subject to increasing anthropogenic transformation, most commonly water pollution, but also other forms of human impact, such as regulation of the river bed and modification of the structure of the banks and bottom of watercourses (Biesiadka, 1972; Biesiadka, Kasprzak, 1977; Kowalik, 1981; Kowalik, Biesiadka, 1981; Cichocka, 1996a; Zawal, Kowalik, 2013; Stryjecki et al., 2018). The effectof human impact on river ecosystems is increasing impoverishment of fauna. Therefore, there is an urgent need to document the state of the Hydrachnidia fauna of rivers, especially natural and unpolluted watercourses. The aim of the study was a detailed faunistic and ecological analysis of the water mites of the small lowland Branew River (central-eastern Poland). The study analysed variation in environmental factors, the species composition of the fauna, dominance structure, species diversity, faunistic similarities, distribution of fauna in the transverse and longitudinal profilesof the river, and synecological structure, as well as the habitat preferences of selected species.

Study area and sites

The Branew is a small lowland river about 22 kilometres long (Geoportal, 2019). It is a right- bank tributary of the Bukowa River. The river begins at the edge of Roztocze with a high-outflow source at an altitude of 245 m (50° 43' 57.95'' N; 22° 33' 33.67'' E), and finishes its course, empty- ing into the Bukowa, in the village of Momoty (50° 43' 57.95'' N; 22° 33' 33.67'' E). The area of the Branew basin is 73.4 km2 (Michalczyk, Wilgat, 1998). The river catchment is narrow, running nearly north to south, with its upper part reaching Roztocze. Apart from the source and the short stretch of the river located in the edge zone of the Western Roztocze mesoregion and the Roztocze macroregion, the river flows through the Biłgoraj Plain mesoregion, which is part of the Sandomierz Basin macroregion (Kondracki, 2014). In the upper reaches, to about half its length, the river flows through open areas.The lower half of the river is in densely forested areas. The Branew River flows through the eastern partof the Janów Forests Landscape Park, crossing it from north to south. The park is located at an altitude of 150–220 m a.s.l., and its relief is not highly diverse. The forests are crossed by numerous parallel valleys of small rivers and streams

101 Robert Stryjecki flowing from the edgesof upland areas. An important element of the hydrographic network of the park is its numerous swamps and peatlands. Many wetlands and fragments of river valleys are occupied by large fish pond complexes situated among forests (Rąkowski et al., 2004). Two sites were selected on the Branew River. Site 1 – in Flisy (50° 39' 33.65'' N; 22° 29' 33.97'' E) A site located about 11 km from the source. This stretch of the river was located in an open area, surrounded by meadows. This part of the river has been transformed by human activity. The transformation involved straightening of the river bed, the partition of the river with a concrete dam, and concrete reinforcement of the bottom for a stretch of a dozen or so metres. The river was 1.5–2.0 m wide and 0.4–0.6 m deep upstream of the dam and 2.0–2.5 m wide and 0.2–0.4 deep downstream. The sediments were diverse, occurring in stretches with a stony, sandy and silty bottom. Below the dam, in the stretch with concrete reinforcement, the bottom consisted of gravel and stones with a small share of sandy sediment. In the shallow water of the lentic zone there were flooded grasses and sedges, as well as isolated specimens of Juncus effusus L. and Equisetum fluviatile L. In the deeper water of the lentic zone, there was abundant Elodea canadensis Michx. In the lotic zone, both upstream and downstream of the dam, Elodea canadensis Michx. occurred in places, but less abundantly that in the lentic zone. Site 2 – at Porytowe Wzgórze (50° 38' 05.27'' N; 22° 27' 58.75'' E) A site located about 15 km from the source. This stretch of the river was surrounded by forest, with numerous meanders. It can be described as natural, without anthropogenic transformations. The river was 3.0–4.0 m wide and 0.1–1.0 m deep. Sandy sediments were dominant over most of the transverse profile of the river, giving way to sandy and silty sediments further from the central part of the river. Silty sediments were dominant by the shores and in pools. There was also a stretch with a stony bottom at this site. Sparganium erectum L. em. RCHB. s. s. grew in the marginal pools and to some extent midstream, forming large clumps. Elodea canadensis was present as well. The banks were covered with flooded grasses – Poa palustris L. and Phalaris arundinacea L.

Methods and material

Field research The field research was conducted from April to November 1996 and from March to October 1997. Samples were taken once a month. At each, site samples were taken from two zones of the river: the lotic zone (in the middle of the river) and the lentic zone (at the riverbank). Sampling was done with a hand net. The net had a round frame of 0.25 m in diameter and 250 µm mesh size. A single sample was taken over a distance of about 10 m. A total of 64 samples were collected. The material comprised a total of 2,785 individuals. The material collected in the field was transported to the laboratory and segregated on white cuvettes. The works used to identify water mites were Viets (1936), Sokolov (1940), Davids et al. (2007), Di Sabatino, Gerecke, Gledhill, Smit (2010), and Gerecke, Gledhill, Pešić, Smit (2016). Species nomenclature and systematics were adopted according to Davids et al. (2007), Di Sabatino et al. (2010) and Gerecke et al. (2016). Allocation of species to synecological groups was based on literature data (Smit, van der Hammen, 2000; Davids et al., 2007; Di Sabatino et al., 2010; Gerecke et al., 2016), taking into

102 Water mites (Acari: Hydrachnidia) of the Branew River account the specific characteristicsof Poland (Biesiadka, 2008) and the region under investigation (Kowalik, 1984). During the collection of hydrobiological samples, the basic physical and chemical indicators of the water were measured: temperature (°C), pH, electrolytic conductivity (μS/cm), dissolved oxygen (mg O2/l), and water saturation with oxygen (%). The measurements were made using a Slandi kit (TM204 thermometer, PH204 pH meter and CM204 conductivity meter) and an Elmetron CX401 multifunction meter. The water current was determined by the floating object method, by measuring the object’s flow time over a distance of 10 m.

Statistical analyses Descriptive statistics (sums, means, range, and standard deviation) were calculated using PAST ver. 3.16/2019 software (Hammer, Harper, Ryan, 2001). The software was also used to calculate the Shannon-Wiener index (H’) and to perform correspondence analysis (CA). Analyses of quantitative faunistic similarities based on the Bray-Curtis formula were carried out using BioDiveristy Pro ver. 2 software (McAleece, Gage, Lambshead, Paterson, 1997). Similarity dendrograms were generated using BioDiveristy Pro software. Group Average was used to create clusters. The normality of the data distribution was checked by the Shapiro-Wilk test. The data were tested for homogeneity of variance using Levene’s test. The t-test was used to compare two independent samples when data had a normal distribution and the variance was homogenous. The Mann-Whitney U test (Z) was used to compare two independent samples when data did not have a normal distribution. The Spearman correlation coefficient (RS) was used to determine the relationship between parameters. All tests were carried out in Statistica 13.1 software. The statistical significance level was set at p = 0.05.

Results

Abiotic environmental parameters The water temperature in the river during the study period ranged from 2.7 to 18.1°C. Both extreme values were found at site 2 (Table 1). The average water temperature was higher at site

2 than at site 1, but the differences between sites were not statistically significant (t30 = –0.6397, p = 0.5171). Water pH ranged from 6.20 to 8.59 (Table 1). Both extreme values were recorded at site 1. The average value of this parameter was slightly higher at site 1 (7.75) than at site 2 (7.25).

The differences were not statistically significant (t29 = 1.2038, p = 0.2383). Electrolytic conductivity during the study ranged from 239 to 970 μS/cm. Both extreme values were found at site 1 (Table 1). Electrolytic conductivity was the only parameter for which the differences between sites were statistically significant (t31 = 2.3287, p = 0.0265). Markedly higher average electrolytic conductivity was recorded at site 1 (508 μS/cm vs 384 μS/cm at site 2). It should be emphasized that this parameter was highly variable during the study at both site 1 and site 2 (±SD 181.31 and ±SD 114.23, respectively). Better oxygen conditions prevailed at site 1 (Table 1), with somewhat higher oxygen content in the water (on average 8.95 mg O2/l vs 8.69 mg O2/l at site 2) and higher oxygen saturation (86.1% vs 80.7% at site 2). The differences in dissolved oxygen content and oxygen saturation were not statistically significant (t23 = 0.3235, p = 0.7491; t23 = 0.8387, p = 0.4102). Water current values ranged from 0.26 to 0.55 m/s (Table 1). Statistically faster water flow was

103 Robert Stryjecki recorded at site 2 (on average 0.33 m/s vs 0.38 m/s at site 1). The differences in water current between sites were not statistically significant (Z = 1.8112, p = 0.0701).

Table 1. Values of analyzed environmental parameters (range; mean; ±SD)

Parameter Site 1 Site 2 2.8–16.4; 2.7–18.1; Temperature (°C) 10.6; ±4.32 11.5; ±4.23 6.20–8.59; 7.26–8.24; pH 7.75; ±0.54 7.55; ±0.34 239–970; 251–695; Electrolytic conductivity (μS/cm) 508a; ±181.31 384b; ±114.23 5.70–12.10; 5.90–12.40; Dissolved oxygen (mg O /l) 2 8.95; ±2.00 8.69; ±1.88 55.0–117.5; 54.0–100.4; Water saturation with oxygen (%) 86.1; ±17.95 80.7; ±14.37 0.28–0.55; 0.26–0.55; Water current (m/s) 0.38; ±0.08 0.33; ±0.07 a, b – the differences in the values between particular sites were statistically significant.

Water mite fauna General characteristics, faunistic similarities and species diversity A total of 2,785 individuals belonging to 32 species, 18 genera and 12 families were caught (Table 2). The dominant species in the material (dominance >5%) were Hygrobates setosus (18.8%), Lebertia rivulorum K. Viets, 1933 (16.1%), Sperchon clupeifer Piersig, 1896 (13.3%), Lebertia inaequalis Koch, 1837 (12.9%), L. fimbriata Thor, 1899 (11.5%) and L. oblonga Koenike, 1911 (10.3%). More individuals (1554) were caught at site 1 (Table 2), but the difference in the numbers of individuals caught at the two sites was not statistically significant (Z = 1.8322, p = 0.0669). Far more species (31) were caught at site 2 (Table 2). A statistically significant correlation was found between the number of individuals caught and the number of species recorded (RS = 0.69, p < 0.05). More individuals and species were recorded in the lentic zone of the river (1494 ind., 27 sp.), than in the lotic zone (1291 ind., 25 sp.), but the differences in the numbers of individuals caught in the two zones were not statistically significant (Z = 0.4541, p = 0.6475). The dominant species in the lentic zone were Hygrobates setosus (26.9%), Sperchon clupeifer (12.9%), Lebertia inaequalis (11.5%), L. fimbriata (11.2%), L. rivulorum (8.8%), L. oblonga (7.5%) and Forelia variegator (6.6%). The dominant species in the lotic zone were Lebertia rivulorum (24.6%), L. inaequalis (14.5%), Sperchon clupeifer (13.6%), Lebertia oblonga (13.4%), L. fimbriata(13.1%) and Hygrobates setosus (9.3%). The fauna of the two sites was 36.3% similar, whereas the water mite communities of the lotic zone and the lentic zone of the river were 70.1% similar. Within each site, the similarity of Hydrachnidia communities of the lotic and lentic zones was nearly identical: 67.57% at site 1 and 67.60% at site 2 (Figure 1).

104 Water mites (Acari: Hydrachnidia) of the Branew River

Table 2. Qualitative and quantitative composition of water mite fauna found in the Branew River. SG – synecological group: Rb – rheobionts, Rp – rheophiles, St – stagnobionts and stagnophiles, S/R – species occurring both in running and stagnant waters, Cf – crenophiles; le – lentic zone of the river, lo – lotic zone of the river, To – total at the site

Site 1 Site 2 In the River No. Species SG le lo To le lo To le lo To 1. Hydrachna globosa (De Geer) St 2 2 2 2 2. Hydrodroma torrenticola (Walt.) Rp 1 1 1 1 3. Hydryphantes placationis Thon St 1 1 1 1 4. Hydryphantes planus Thon St 2 2 2 2 5. Lebertia fimbriataThor Rp 26 41 67 124 125 249 150 166 316 6. L. oblonga Koen. Rp 9 15 24 102 155 257 111 170 281 7. L. rivulorum Viets Rb 115 293 408 14 19 33 129 312 441 8. L. inaequalis (Koch) Rp 105 143 248 64 41 105 169 184 353 9. L. insignis Neum. Rp 5 5 1 2 3 1 7 8 10. L. pilosa Maglio Rp 8 2 10 115 18 56 46 20 66 11. L. porosa Thor Rp 1 1 1 1 – Lebertia sp. (deutonymphs) – 3 4 7 3 4 7 6 8 14 12. Sperchon clupeifer Piers. Rb 177 147 324 14 25 39 191 172 363 13. S. setiger Thor Rb 1 1 2 1 3 3 1 4 – Sperchon sp. (deutonymphs) – 7 3 10 7 3 10 14. Sperchonopsis verrucosa (Protz) Rb 3 3 6 1 – 1 4 3 7 15. Teutonia cometes (Koch) S/R 5 – 5 5 5 16. Torrenticola amplexa (Koen.) Rb 1 1 1 1 17. Aturus scaber Kram. Rb 1 1 1 1 18. Parabrachypoda modesta (Koen.) Rp 3 2 5 3 2 5 19. P. montii (Maglio) Rp 18 3 21 18 3 21 20. Atractides distans (Viets) Rb 12 3 15 5 5 10 17 8 25 21. A. nodipalpis Thor Rb 10 26 36 5 10 15 15 36 51 22. A. ovalis Koen. S/R – 1 1 1 1 – Atractides sp. (deutonymphs) – 1 2 3 1 2 3 23. Hygrobates calliger Piers. Rb 1 2 3 1 2 3 24. H. fluviatilis (Ström) Rb 20 12 32 5 6 11 25 18 43 25. H. longipalpis (Herm.) S/R 9 1 10 9 1 10 26. H. setosus Bess. Rp 261 85 346 135 33 168 396 118 514 – Hygrobates sp. (deutonymphs) – 3 1 4 4 7 11 7 8 15 27. Forelia variegator (Koch) S/R 9 1 10 88 15 103 97 16 113 28. Nautarachna crassa (Koen.) Rp 2 – 2 2 2 – Piona sp. (deutonymphs) – 1 – 1 1 1 29. Tiphys ornatus Koch St 2 – 2 2 2 30. Wettina podagrica (Koch) Cf 4 1 5 4 1 5 31. Mideopsis crassipes Soar Rp 55 21 76 55 21 76 32. M. roztoczensis Bies. et Kow. Rp 13 – 13 13 13 – Mideopsis sp. (deutonymphs) – 1 4 5 1 4 5 Total individuals 769 785 1,554 725 506 1,231 1,494 1,291 2,785 Total species 13 14 15 27 23 31 27 25 32

105 Robert Stryjecki

Figure 1. Faunistic similarities between zones at particular sites in the Branew River. 1–2 – sites, lentic – lentic zone, lotic – lotic zone

The total species diversity of the Hydrachnidia communities of the Branew River was H’ = 2.33. Higher species diversity was found at site 2 (H’ = 2.37) than at site 1 (H’ = 1.83). Higher species diversity was recorded in the lentic zone than in the lotic zone of the river, both on the scale of the entire river and at each of the sites (Figure 2).

Figure 2. Shannon-Wiener index values at particular sites and lentic and lotic zones in the Branew River

Synecological structure of the fauna Rheophiles were the largest synecological group in the Branew River (76.4%, 13 sp.), followed by rheobionts (18.5%, 10 sp.). Four species classified as occurring in both running and stagnant waters accounted for only 4.7% of the collected fauna. The other two synecological elements, stagnobionts and stagnophiles (4 species) and crenophiles (1 species), made up a very

106 Water mites (Acari: Hydrachnidia) of the Branew River small proportion of the fauna (0.3% and 0.2%, respectively). Differences were noted in the synecological structure of the fauna between the lentic and lotic zones of the river. In the lentic zone, there was a noticeable (7.5%) share of taxa classified as species occurring both in running and stagnant waters, while the proportion of these species in the lotic zone was negligible (Figure 3). Water mites characteristic of running water bodies (rheobionts and rheophiles) together accounted for 91.7% of the fauna in the lentic zone, while in the lotic zone they made up as much as 98.5% of the collected fauna (Figure 3). Representatives of other synecological groups were caught in very small numbers or not at all in the central part of the river (Table 2).

Figure 3. Quantitative synecological structure of water mite fauna in the lentic and lotic zone in the Branew River

Occurrence and distribution of water mites in the transverse profile of the river For the seven most abundant species (>100 specimens), an analysis was performed of the distribution in the transverse profile of the river. The species most associated with the lotic zone was Lebertia rivulorum, with as many as 70.7% of individuals caught in the central part of the river (Figure 4). Another species that preferred current environments was L. oblonga (60.5% of specimens caught in the lotic zone). Another two species present in high numbers in the lotic zone were L. fimbriata and L. inaequalis, but they were caught in only slightly higher numbers in the central part of the river than by the banks (52.5% and 52.1%, respectively). The species most associated with the lentic zone was Forelia variegator (85.8% of individuals were caught in this zone; Figure 4). Another species with clear preferences for the littoral zones of the river was Hygrobates setosus; 77.1% of individuals of this species were caught in the marginal pools. Another species that was somewhat more abundant outside the current zone was Sperchon clupeifer, but this species cannot be considered to be associated with the lentic zone because its proportion was only slightly higher there (52.6%) than in the lotic zone.

107 Robert Stryjecki

Figure 4. Percentage share of the most numerous species in lentic and lotic zone in the Branew River

Occurrence and distribution of water mites in the investigated part of the river Correspondence analysis was performed for the seven most abundant species (>100 specimens), taking into account the distribution in both the transverse and longitudinal profilesof the river. Lebertia rivulorum was caught in the upper course of the river (site 1), in the lotic zone, on a bottom of stones and gravel with an admixture of sandy sediment (Figure 5). Another characteristic species of this part of the river was Sperchon clupeifer, with uniform distribution in the transverse profile of the river (Figures 4 and 5). Two species, Hygrobates setosus and Forelia variegator, were clearly associated with the lentic zone of the river (Figure 5), but Hygrobates setosus was caught mainly in the upper course of the river (site 1, silty bottom with Elodea canadensis), while as much as 91.5% of the population of Forelia variegator was caught in the lower course of the river (site 2, bottom of silt, silt and sand, and sand, with dominance of Sparganium erectum and some Elodea canadensis). Lebertia fimbriata and L. oblonga were also characteristic of the lower course of the river (Figure 5). Lebertia oblonga was associated with the lotic zone of the lower course of the river (site 2, sediments of sand or sand and silt with isolated Sparganium erectum plants and small patches of Elodea canadensis), while L. fimbriata showed a more even distribution in the transverse profileof the river, with only a slight preference for the current environment (Figures 4, 5). More specimens of L. inaequalis were caught in the upper course of the river and in the lotic zone, but this species was also found in high numbers at site 2 and in the lentic zone (Table 2, Figures 4, 5).

108 Water mites (Acari: Hydrachnidia) of the Branew River

Figure 5. Correspondence analysis (CA) showing the occurrence of the most numerous species in the Branew River. 1–2 – sites, le – lentic zone, lo – lotic zone, For_var Forelia– variegator, Hyg_set – Hygrobates setosus, Leb_fim –Lebertia fimbriata, Leb_ina – L. inaequalis, Leb_obl – L. oblonga, Leb_riv – L. rivulorum, Spe_clu – Sperchon clupeifer

Discussion

In the Hydrachnidia fauna of the Branew River, there are a few characteristic elements that distinguish the water mite assemblages of this river from other Polish rivers. The first was the very high abundance of Lebertia rivulorum. Apart from the Branew, this species has also been recorded in other rivers of the Janów Forests Landscape Park (Stryjecki, 2002; Stryjecki et al., 2018), as well as in other rivers of the Biłgoraj Plain (Zawal, Kowalik, 2013), but in much smaller numbers than in the Branew River. In rivers situated in neighbouring geographic regions, in some cases in close proximity to the Biłgoraj Plain, L. rivulorum has been caught in very small numbers (Kowalik, Biesiadka, 1981; Kowalik et al., 2014) or not at all (Kowalik, 1981; Stryjecki, 2009, 2010; Stryjecki et al., 2012; Stryjecki, Kowalczyk-Pecka, 2013b; Biesiadka et al., 2015). In rivers of other parts of Poland, this species has been recorded in small or very small numbers (Biesiadka, 1970, 1972; Zawal et al., 2017) or not at all (Biesiadka, 1979; Cichocka, 1996a, b, 2006; Zawal, Sadanowicz, 2012). The analysis indicates that the abundance of L. rivulorum in the rivers of the Sandomierz Basin macroregion and the Biłgoraj Plain mesoregion is a regional feature. Lebertia rivulorum has been recorded in small numbers in European rivers and streams (van der Hammen, Smit, 1996; Martin, Speth, 1996; Martin, 1997; Biesiadka, Cichocka, Moroz, 2004; Smit, Hop, Munts, 2008), or not recorded at all (Gledhill, 1973; Martin, 1996; Gerecke, 2002). The material collected during the study shows that the population of this species in the Branew River was not only the highest in Poland, but in all of Europe. Lebertia rivulorum is considered a rhithrobiont (Gerecke, 2009). It prefers an environment of mosses (Dittmar, 1955) and Alnus roots (Martin, Spetch, 1996). In the Branew, this species was caught primarily in the upper stretches of the river, on a bottom of gravel

109 Robert Stryjecki and stones with a small amount of sandy sediment, sparsely covered with Elodea canadensis. The results contribute new data on the habitat preferences of this species. Another characteristic element of the Hydrachnidia communities of the Branew River, which distinguishes the river from other Polish rivers, was the very high abundance of Lebertia inaequalis. This species has also been found in high numbers in other rivers of the Janów Forests Landscape Park (Stryjecki, 2002; Stryjecki et al., 2018) and in other rivers of the Biłgoraj Plain (Zawal, Kowalik, 2013). In the neighbouring macroregion of Roztocze, L. inaequalis has been much less abundant than in the rivers of the Sandomierz Basin (Kowalik, 1981; Stryjecki, Kowalczyk-Pecka, 2013b; Kowalik et al., 2014; Biesiadka et al., 2015). Lebertia inaequalis has been recorded in other lowland rivers in Poland (Biesiadka, 1972; Cichocka, 1996a, 2006; Zawal, Sadanowicz, 2012; Zawal et al., 2017), but in none of these has it reached such high abundance and dominance as in the Branew and other rivers of the Biłgoraj Plain. Another distinctive feature was the high abundance of Lebertia oblonga. At just two sites in the small Branew, far more individuals were caught than at a greater number of sites in larger rivers of the Janów Forests Landscape Park (Stryjecki et al., 2018), the Biłgoraj Plain (Zawal, Kowalik, 2013), neighbouring Roztocze (Stryjecki, Kowalczyk-Pecka, 2013b; Kowalik et al., 2014; Biesiadka et al., 2015), and rivers located in other parts of Poland (Zawal, Sadanowicz, 2012; Zawal et al., 2017). In general, the high proportion of species of the genus Lebertia was a characteristic feature of the Hydrachnidia communities of the Branew River, distinguishing its fauna from that of other Polish rivers and other rivers of the region. Species of this genus accounted for as much as 53.1% of the collected material. Among species of the genus Lebertia that were caught in high numbers in the Branew River, two – L. oblonga and L. pilosa Maglio, 1924 – are rare in Europe (Gerecke, 2009). As mentioned above, L. oblonga has been recorded in other Polish rivers, but never in such abundance as in the Branew. This species has been found in small numbers in the rivers and streams of other parts of Europe (Martin, 1997; Smit et al., 2015), or not at all (Martin, 1996; Martin, Speth, 1996; Gerecke, 2002; Biesiadka et al., 2004). The material collected during the study shows that the population of this species in the Branew River was the largest not only in Poland, but over its entire range of occurrence. Lebertia oblonga is found in high-order lowland streams, but also in clear lakes, often on sandy bottoms (Gerecke, 2009; Smit et al., 2015). In the Branew River, L. oblonga was caught primarily in the current zone, on a bottom of sand or sand and silt, with a small share of aquatic vegetation, in the lower course of the river. The data confirm the habitat preferences of this species known from the literature. Lebertia pilosa is a rare species, found only in small populations. Its distribution includes Europe, but only scattered records are known (Gerecke, 2009). In Poland, L. pilosa has been found in rivers in the central-eastern part of the country, sometimes in fairly high numbers (Kowalik, 1981; Kowalik, Biesiadka, 1981; Zawal, Kowalik, 2013; Biesiadka et al., 2015; Stryjecki et al., 2018). In other Polish rivers, the species has been found in low numbers (Cichocka, 1996a, 2006) or not at all (Zawal, Sadanowicz, 2012; Zawal et al., 2017). The fact that 66 L. pilosa individuals were found in the Branew, a small lowland river, is indicative of the faunistic value of this river. According to Kowalik (1981), L. pilosa prefers environments with a weaker current on bottoms covered with vegetation. In the Branew, it was found mainly in the littoral zone in the lower course of the river, in places with sediments of silt or sand and silt, overgrown with abundant aquatic vegetation, which confirms the previously reported habitat preferences of the species. High similarity was noted between the fauna of the lotic and lentic zones of the river, and much lower similarity between sites. The results indicate intensive migration of species in the

110 Water mites (Acari: Hydrachnidia) of the Branew River transverse profileof the river and less migration in the longitudinal profile. The Branew River is a small watercourse; its width at the study sites ranged from 1.5 to 4.0 m, which was undoubtedly conducive to the exchange of species between the littoral zone and the central zone of the river. In the main river of the Janów Forests Landscape Park, i.e. the Bukowa River, Stryjecki et al. (2018) found high faunistic similarity between the lotic and lentic zones in the upper course of the river within the same site, but in the lower course of the river the fauna was grouped within the lotic or lentic zone of the various sites. In the Krąpiel River, Zawal et al. (2017) noted the highest faunistic similarity between similar habitats of different sites, especially in the lower courseof the river. The Branew River is too small and short for such a pattern of faunistic similarities to be observed in its lower course. Human impact and the transformation of lotic ecosystems causes changes in water mite fauna: rheophilic species, with narrow tolerance for environmental factors, vanish and are gradually replaced by species with a wider ecological valence, which are frequently found in standing water bodies as well (Biesiadka, 1972; Martin, 1996; van der Hammen, Smit, 1996). The largest synecological group in the Branew River was rheophiles and rheobionts. These two groups are usually dominant in rivers, especially natural rivers or those subject to little human impact (Biesiadka, 1970; Cichocka, 1996a; Zawal et al., 2017; Stryjecki et al., 2018). Dominance of rheobionts and rheophiles has also been noted in other lowland rivers in Poland (Biesiadka, 1970; Cichocka, 1996b, 2006; Zawal, Sadanowicz, 2012), as well as in upland and lowland rivers of central-eastern Poland (Kowalik, 1981; Stryjecki, Kowalczyk-Pecka, 2013b; Zawal, Kowalik, 2013; Kowalik et al., 2014; Biesiadka et al., 2015). The very high quantitative share of rheophiles and rheobionts in the fauna of the Branew River is indicative of its natural character. The values of the physicochemical parameters confirm the good water quality of the river. The most abundant species in the Branew River, Hygrobates setosus, was caught in much higher numbers in the littoral zone than in the central part of the river. Discussion on the habitat preferences of this species is impeded by the fact that many publications on flowing water bodies identify this species as H. nigromaculatus, a sister species occurring in lakes. The use of incorrect nomenclature in previous works is due to the fact that the river species (H. setosus) was not distinguished from the lake species (H. nigromaculatus) until relatively recently (Martin et al., 2010). Hence the species reported as H. nigromaculatus in many papers on lotic ecosystems was most likely H. setosus. This is suggested by the abundance of this species in the flowing water bodies discussed by the authors and its classification in these studies as a rheophile (Biesiadka, 1979; Cichocka, 1996a; Martin, 1996; Kowalik et al., 2014), whereas H. nigromaculatus is a typical lake species (Martin et al., 2010). In the Branew River, H. setosus displayed a very clear preference for the lentic zone of the river. In other Polish rivers, H. setosus has also been found mainly in marginal pools and habitats with slow current (Kowalik, 1981; Cichocka 1996a; Zawal, Sadanowicz, 2012; Zawal et al., 2017; Stryjecki et al., 2018). In the Pasłęka River, H. setosus clearly preferred pools with a sandy bottom without vegetation (Cichocka, 1996a). This species is much more frequently reported as characteristic of pool areas of rivers with organic (mud) sediments (Biesiadka, 1979; Martin, 1996), often with rich aquatic vegetation (Kowalik, 1981). A great deal about the preferences of this species can be learned from a comparison of the distribution of H. setosus in the Branew River and the larger Bukowa River, which the Branew flows into. At just two sites in the Branew, many more H. setosus individuals (514) were caught than at five sites in the much larger Bukowa River (107 individuals; Stryjecki et al., 2018). In the Bukowa River, sandy sediments were dominant and vegetation was generally sparse. In the Branew River, H. setosus was present in the highest numbers in the lentic zone of the river, where silty sediments

111 Robert Stryjecki and abundant aquatic vegetation were dominant (mainly Elodea canadensis, but also Sparganium erectum). Comparison of the populations from the Bukowa and Branew Rivers indicates that sandy sediment and sparse aquatic vegetation are not suitable habitat conditions for the formation of such large populations of H. setosus. Suitable conditions are found in places with slow water flow and a silty bottom with abundant aquatic plants, which additionally slow down the water current. Documentation of the habitat preferences of H. setosus is important because it enables an increasingly precise understanding of these preferences. At the anthropogenically transformed site 1, species diversity was much lower than at the natural site 2. According to Odum (1982), low biodiversity is characteristic of unstable biocoenoses subject to seasonal or periodic disturbances caused by humans or nature. The typical response of a biocoenosis to environmental stress is a decrease in the number of species that are represented in small numbers, with a simultaneous increase in the dominance of species with high tolerance to stress. In the case of site 1, the environmental stress was anthropogenic transformation of that stretch of the river. The low value of the Shannon-Wiener index at this site was influenced by both of its components: a small number of species (14) and an uneven distribution of dominance (very high abundance of Lebertia rivulorum, Hygrobates setosus, Sperchon clupeifer and Lebertia inaequalis). Straightening of the river bed, erection of a concrete dam and concrete reinforcement of the bottom in the investigated stretch of the river led to habitat impoverishment, which trans- lated into species impoverishment. Few species were caught at site 1, with especially few species characteristic of standing water bodies, which were collected in the expansive marginal pools in the meanders at the natural site 2 (e.g. Hydrachna globosa (De Geer, 1778), Hydryphantes placationis Thon, 1899, and Tiphys ornatus Koch, 1836). The artificial, man-made bottomof stones and gravel, with a small admixture of sandy sediment, proved suitable for Lebertia rivulorum, which had a very large population here. These habitat conditions also proved favourable for Sperchon clupeifer, which has not been found in any other river of the Janów Forests Landscape Park in such high numbers as in this anthropogenically transformed stretch of river. The concrete-reinforced bottom and substrate of stones and gravel with an admixture of sandy sediment was a substitute for the natural habitat conditions preferred by this species, i.e. a substrate of stones and sand (Di Sabatino, Gerecke, Martin, 2000). Human impact involving regulation of the river channel and changes in the bottom structure usually lead to habitat degradation and impoverishment of water mite fauna (Biesiadka, 1972; Martin, 1996; van der Hammen, Smit, 1996; Stryjecki et al., 2018). The results confirm literature data describing the negative effect of such transformations on the Hydrachnidia communities of rivers.

References

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Cite as: Stryjecki, R. (2019). A faunistic and ecological characterization of the water mites (Acari: Hydrachnidia) of the Branew River (central-eastern Poland). Acta Biologica, 26, 99–115. DOI: 10.18276/ ab.2019.26-09. #1#

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Acta Biologica 26/2019 | www.wnus.edu.pl/ab | DOI: 10.18276/ab.2019.26-10 | strony 117–126

Scanning electron microscopic observation of Acarothrix grandocularis (Acari, Halacaridae) and notes on the species of the genus Acarothrix

Tapas Chatterjee

Crescent International School, Bario, Govindpur, Dhanbad 828109, Jharkhand, India

Corresponding author, e-mail: [email protected]

Keywords Acarothrix grandocularis, SEM, Distribution Abstract Scanning electron microscopic observations of some characters of Acarothrix grandocularis Chatterjee, Marshall, Guru, Ingole, Pešić 2012 is presented. Distribution and characters of species belonging to the genus Acarothrix are also discussed.

Cechy Acarothrix grandocularis (Acari, Halacaridae) widoczne dzięki zastosowaniu mikroskopu skaningowego na tle rodzaju Acarothrix Słowa kluczowe Acarothrix grandocularis, SEM, rozmieszczenie Streszczenie Artykuł prezentuje cechy Acarothrix grandocularis Chatterjee, Marshall, Guru, Ingole, Pešić 2012 – widoczne dzięki zastosowaniu mikroskopu skaningowego. Dyskutowane są również cechy i rozmieszczenie gatunków należących do rodzaju Acarothrix.

Introduction

Acarothrix grandocularis Chatterjee, Marshall, Guru, Ingole and Pešić (2012) was first described from India and Brunei Darussalam among algal turf growing on pneumatophores of mangroves in estuarine habitats (Chatterjee et al., 2012). Bartsch (2015) reported this species from Kranji, Singapore among green and red algae on trunks of mangrove trees. In the present paper some morphological characters of A. grandocularis is studied in detail, based on scanning electron microscopic observation of specimens from Goa, India. Distribution and characters of species belonging to the genus Acarothrix are also provided.

117 Tapas Chatterjee

Material and Methods

Specimens were collected from Chorao Island (North Goa), Virnoda Pernem (North Goa), Chicalim Vasco (South Goa) and Chinchinim (South Goa) among algal turf growing on pneumatophores of mangroves or in mud flat associated with mangroves. Specimens for scanning electron microscopy (SEM) were prefixed overnight at 4°C in 2.5% glutaraldehyde, followed by post fixation in 2% cold osmium tetroxide. After dehydration through a graded series of ethanol (50–100% at 10% interval) for 30 minutes each, the material was critical-point dried, and coated with a platinum-palladium mix in a high evaporator, and then examined with a scanning electron microscope. The following abbreviations are used in the text and figure legends: AD, anterior dorsal plate; AE, anterior epimeral plate; ds1-5, dorsal setae 1–5 on the idiosoma; GA, genitoanal plate; GO, genital opening; OC, ocular plate(s); PAS, parambulacral seta(e); PD, posterior dorsal plate; PGS, perigenital setae; P1-4, first to fourth palpal segment; SGS, subgenital setae.

Study area: Chorao Island (North Goa), west coast of India: Latitude 15° 30' 45.74'' N, Longitude 73° 52' 11.25'' E. The Chorao Island is situated 5 kms from Panaji. Island present in Mandovi river, water is brackish in nature and salinity from 3–7‰. Samples collected from algal turf associated with pneumatophores of Avicennia sp, Rhizophora sp. Chicalim Vasco (South Goa), west coast of India: Latitude 15° 24' 20.49'' N, Longitude 73° 53' 18.97'' E. Samples collected from algal turf associated with pneumatophores of Avicennia and Rhizophora sp. Samples also collected from mud flat associated with mangroves. Virnoda Pernem (North Goa), west coast of India: Latitude 15° 40' 13.85'' N, Longitude 73° 43' 22.19''. Samples collected from algal turf associated with pneumatophores of Rhizophora sp. Chinchinim (South Goa), west coast of India: Chinchinim is located between Lat: 15° 12' N. Long: 73° 58' E and 15.20° N 73.97° E. Samples collected from mud flat in mangrove area. Avicennia sp dominated.

Results and Discussion

Acarothrix grandocularis Chatterjee et al. 2012 Acarothrix grandocularis Chatterjee et al. (2012, pp. 542–546, figs. 1A-D, 2A-F, 3A-D); Bartsch (2015, 100–102, figs. 2H-O).

Description: The original description of this species is given in Chatterjee et al. (2012) based on the specimens collected from Goa, India and Brunei Darussalam. Some characters referred in that paper are described in more details according to present SEM study based on the specimens collected from Goa, India. AD, OC and PD are separate (Figure 1A). Areolae and costae on dorsal plates slightly raised with porose panel (Figures 1F, 2A); remainder of plates panelled (2B). AD with one anterior and two posterior areolae; 1st pair of gland pores inserted near anterolateral edge of posterior areolae; posterior margin of AD triangular. OC elongate, setae ds2 on OC (Figure 1D, E). PD with a pair of longitudinal porose costae, 2 porose panels wide (Figures 1F, 2A); setae ds3–ds5 on PD (2C, D). AE, PE and GA separate (Figure 1B, C). AE almost smooth (Figure 2E) with 3 pairs of ventral setae and a pair of epimeral pores, epimeral pore shown in Figure 2F. Three pairs of PGS present. Pair of SGS located at the anterior end of genital sclerites (Figure 3A).

118 Scanning electron microscopic observation of Acarothrix grandocularis (Acari, Halacaridae)...

Figure 1. Acarothrix grandocularis Chatterjee et al. 2012, SEM figures, female. A. Idiosoma dorsal; B, C. Idiosoma ventral; D. Magnified view of parts of AD, OC and PD; E. OC and PD; F. Part of costa and panels on PD.

119 Tapas Chatterjee

Figure 2. Acarothrix grandocularis Chatterjee et al. 2012, SEM figures, female. A. Part of costa on PD; B. Panels between two costae on PD; C. seta ds3 on PD; D. Seta ds4 on PD; E. Part of AE; F. Epimeral pore on AE.

120 Scanning electron microscopic observation of Acarothrix grandocularis (Acari, Halacaridae)...

Figure 3. Acarothrix grandocularis Chatterjee et al. 2012, SEM figures, female. A. GO of female; B. Gnathosoma ventral view; C. Gnathosoma ventrolateral view; D. Anterior part of gnathosoma and palp; E. Part of tibia I and tarsus I; F. Part of tibia II and tarsus II (arrow indicating pectinate seta on tibia).

121 Tapas Chatterjee

Figure 4. Acarothrix grandocularis Chatterjee et al. 2012, SEM figures, female. A, Part of tibia II and tarsus II (arrow indicating pectinate seta on tibia); B. Anterior part of tarsus II (arrow indicating 1., 2: PAS; 3: solenidion); C. Anterior part of tarsus II (arrow indicating PAS); D, E. Part of tibia and tarsus III (arrow indicating pectinate seta on tibia); D. Tip of tarsus III.

122 Scanning electron microscopic observation of Acarothrix grandocularis (Acari, Halacaridae)...

Rostrum triangular, tip of rostrum not surpassing the anterior end of P2. P1 and P3 without any setae; P2 with 1 dorsal seta; P4 with 3 long proximal setae and 1minute distal seta. Proto and deuto- rostral setae situated at the tip of rostrum, long maxillary setae of rostrum anterior to middle of rostrum, gnathosomal base with a pair of setae (3B, C). Rostral sulcus is extending posteriorly to just beyond the tritorostral setae (Figure 3B, D). Tibiae I–IV with 1-1-1-0 bipectinate ventromedial setae . Pectinate seta shown in figures 3E, F, 4A, D, E. Tarsus I with 3 dorsal setae, 1 solenidion, 2 ventral setae, 2 doublet eupathid PAS (Figure 3E). Tarsus II with 3 dorsal setae, 1 solenidion, 2 single eupathid PAS (4B, C). Tarsus III with 4 dorsal setae and 2 PAS (4F). All tarsi with 2 lateral claws, a small bidentate medial claw, and a carpite; lateral claws smooth ventrally.

Remarks. In India. A. granocularis is found from both pneumatophores turf and mud flat also. The salinity of mangrove area ranges from 3–7‰. A detailed study based on SEM and molecular analysis for specimens from different localities is necessary to reveal variations in this species between the localities.

Notes on species of the genus Acarothrix Acarothrix is a genus of halacarid mites that was proposed by Bartsch (1990) and has A. palustris Bartsch 1990 as the type species. There are five species viz. A. palustris Bartsch (1990), A. longiunguis Bartsch (1997), A. umgenica Procheş (2002), A. ampliata Bartsch (2004) and A. grandocularis Chatterjee et al. (2012) so far recorded under this genus. The genus has been recorded from Southern China, Singapore, northern Australia, south- eastern Africa, Florida and India. All species are known from tropical or warm temperate regions. Table 1 shows detail distribution of the species in Acarothrix along with the habitats and references.

Table 1. Species of Acrothrix: Localities with habitats

Name of the species Locality Oceanic provinces Habitat References

1 2 3 4 5 Acarothrix USA: Gulf of Mexico ATW: Atlantic Ocean, Little Manatee River, Bartsch (2004) ampliata at Tampa Bay, Florida tropical west which empties into Bartsch Tampa Bay 2004 Acarothrix Singapore PTW: Pacific Ocean, Cladophora mat, Bartsch (2006) ampliumeris tropical west Chlorophyta, on Bartsch 2006 muddy and sandy sediments in mangrove area

Singapore: End of Lim PTW: Pacific Ocean, Among green Bartsch (2015) Chu Kang Road tropical west (Cladophorales) and red algae (Catenella sp., Gigartinales) on pneumatophores of Avicennia sp. (Avicenniaceae) mangroves

123 Tapas Chatterjee

1 2 3 4 5 Acarothrix Brunei Darussalam: PTW: Pacific Ocean, Algal turf growing Chatterjee et al. (2012) grandocularis Batu Marang tropical west on Rhizophora sp. Chatterjee, pneumatophores Marshall, Guru, Ingole, Pešić 2012 India: Chorao Island, ITE: Indian Ocean, Algal turf growing on Chatterjee et al. North Goa tropical east Avicennia, Rhizophora (2012); Present report pneumatophores

Singapore: Kranji PTW: Pacific Ocean, landward edge of Bartsch (2015) tropical west mangrove area, green and red algae on trunk, high water edge

India: Chicalim Vasco ITE: Indian Ocean, Algal turf growing Present report (South Goa) tropical east on pneumatophores of mangroves; mud flat associated with mangroves

India: Virnoda Pernem ITE: Indian Ocean, Algal turf growing on Present report (North Goa): tropical east pneumatophores of mangroves;

India: Chinchinim ITE: Indian Ocean, Mud flat on the Present report (South Goa) tropical east mangrove area. Acarothrix Australia: Sadgroves PTW: Pacific Ocean, Soft mud from Bartsch (1997) longiunguis Creek, near Darwin, tropical west mangrove area Bartsch 1997 Northern Australia, Acarothrix Hong Kong: southern PTW: Pacific Ocean, Algal turf on salt Bartsch (1990) palustris Bartsch China tropical west marshes and mangrove 1990 flats

Singapore: Pandan PTW: Pacific Ocean, Green algae and Bartsch (2006) River, southern coast tropical west epibiota on Avicennia of Singapore pneumatophores in a rockpool

Singapore: End of PTW: Pacific Ocean, Cladophora mat on Bartsch (2006) Lim Chu Kang Road, tropical west muddy and sandy northern coast of sediment in mangrove Singapore area

India: Chorao Island, ITE: Indian Ocean, From algal turf Chatterjee et al (2013) North Goa tropical east growing on Avicennia pneumatophores Acarothrix South Africa: ITW: Indian Ocean, sediment on Avicennia Procheş et al. (2001) umgenica Procheş Beachwood and tropical west pneumatophores 2002 Bayhead Lagoon, near Durban, KwaZulu-Natal

South Africa: ITW: Indian Ocean, Sediment or algae Procheş (2002) Beachwood tropical west covering the mangroves in Durban, pneumatophores of and Richards Bay, the Avicennia marina KwaZulu-Natal mangrove tree

124 Scanning electron microscopic observation of Acarothrix grandocularis (Acari, Halacaridae)...

All species live in intertidal muddy environments characterized by fluctuating salinities. Four species of Acarothrix are found to be associated with mangroves (Chatterjee, Pfingstl, Pešić, 2018a). Sexual dimorphism is common in many groups of Arthropoda. Sexual dimorphism is also found in some species of halacarid mites (Chatterjee, Guru, 2012). A pair of external genital acetabula is present on genital sclerites in males of the genus Acarothrix, while the external genital acetabula on genital sclerites are absent in the female. A comparison of important characteristics between species in the genus Acrothrix is com- piled in Table 2.

Table 2. Comparison of important characteristics between species in the genus Acarothrix

Acarothrix Acarothrix Acarothrix Acarothrix Acarothrix Acarothrix Characters ampliata ampliumeris grandocularis longiunguis palustris umgenica Male 314–325 Male 278–286 Idiosoma Male 326–340 Male 278–291 Female 294– Female 279– Male 287–322 325–385 length Female 340 Female 291–293 309 294 Posterior end Triangular Triangular Triangular Rounded Rounded Triangular of AD Remnants Cornea on OC Absent Present Present Present Present of cornea Position of Anterior part About middle Posterior part of About middle About middle Posterior part ds1 on AD on AD AD of AD of AD of AD Position of Membranous OC OC OC OC OC ds2 cuticle Position of OC OC PD OC OC OC ds3 Very faint line Present, two Costae on PD Absent Absent Present Absent like porose panel wide Median portion Reticulate Reticulate panels Smooth area Reticulate Reticulate Panels on PD deliculately panels on PD on PD on PD panels on PD panels on PD reticulate Wart on membranous cuticle on Present Absent Absent Absent Present Absent idiosoma dorsal Setae on 3 3 2 3 3 3 basifemur III Bipectinate seta in tibiae I 1-1-1-0 1-1-1-1 1-1-1-0 1-1-1-0 1-1-1-0 1-1-1-? to IV Distance between 0.7 of GO Slightly less 0.7 of GO Equal with 1.8 of Go anterior end 1.1 of GO length length than GO length length GO length length of GO and GA in male

Suctorian and Peritrich ciliate epibionts have been found on several halacarid mites (Chatterjee, Dovgal, Pešić, Zawal, 2018b). Bartsch (2015) reported suctorian ciliate Praethecacineta halacari (Schulz, 1933) on Acarothrix grandocularis from Singapore.

125 Tapas Chatterjee

Acknowledgement Thanks are due to Dr. Martin V. Sorensen, Natural History Museum of Denmark, University of Copenhagen, Denmark for making the SEM photographs; to Dr Mandar Nanajkar, CSIR, National Institute of Oceanography, Dona Paula, Goa-403004, India and Mr. Souhardya Chatterjee for their help to arrange the figure plates.

References

Bartsch, I. (2004). Acarothrix ampliata (Arachnida: Acari: Halacaridae), a new halacarid mite from Flori- da, with notes on external genital acetabula. Species Diversity, 9, 259–267. Bartsch, I. (1990). Acarothrix palustris gen. et spec. nov. (Halacaroidea, Acari), ein Bewohner der Salzwi- esen Südchinas. Zoologischer Anzeiger, 224, 204–210. Bartsch, I. (1997). Copidognathinae (Halacaridae, Acari) from northern Australia; description of four new species. In: J.R. Hanley, G. Caswell, D. Megirian, H.K. Larson (eds.), Proceedings of the Sixth In- ternational Biological Workshop (pp. 231–243). Museums & Art Galleries of Northern Territory, Darwin. Bartsch, I. (2006). Copidognathines (Acari: Halacaridae) in mangroves of Singapore. I. Description of three species. The Raffles Bulletin of Zoology, 54, 83–92. Bartsch, I. (2015). Halacaridae (Acari) amongst the epiflora fauna on trunk, branches, roots and pneumatophores on the coast of Singapore: a survey. Raffles Bulletin of Zoology, 31 (supplement), 96–138. Chatterjee, T., Dovgal, I., Pešić, V., Zawal, A. (2018b). A checklist of epibiont suctorian and peritrich ciliates on halacarid and hydrachnid mites (Acari: Halacaridae and Hydrachnidia). Zootaxa, 4457 (3), 415–430. Chatterjee, T., Guru, B.C. (2012). Sexual dimorphism in halacarid mites (Acari, Halacaridae): An updated overview. Acta Biologica, 19, 99–12. Chatterjee, T., Guru, B.C., Sorensen, M.V. (2013). Report of Acarothrix palustris Bartsch (Acari: Halacari- dae) from the Indian Ocean, Acta Biologica, 20, 17–26. Chatterjee, T., Marshall, D.J., Guru, B.C., Ingole, B., Pešić, V. (2012). A new species of the genus Acaro- thrix (Acari, Halacaridae) from Brunei Darussalam and India. Cahiers De Biologie Marine, 53 (4), 541–546. Chatterjee, T., Pfingstl, T., Pešić, V. (2018a). A checklist of marine littoral mites (Acari) associated with mangroves. Zootaxa, 4442 (2), 221–240. Procheş, Ş. (2002). New species of Copidognathinae (Acari: Halacaridae) from southern Africa. Journal of Natural History, 36, 999–1007. Procheş, Ş., Marshall, D.J., Ugrasen, K., Ramcharan, A. (2001). Mangrove pneumatophores arthropod assemblages. Journal of the Marine Biological Association, 81, 545–552.

Cite as: Chatterjee, T. (2019). Scanning electron microscopic observation of Acarothrix grandocularis (Acari, Halacaridae) and notes on the species of the genus Acarothrix. Acta Biologica, 26, 117–126. DOI: 10.18276/ab.2019.26-10. #1#

126 #0#

Acta Biologica 26/2019 | www.wnus.edu.pl/ab | DOI: 10.18276/ab.2019.26-11 | strony 127–132

Report of Rhombognathus scutulatus (Acari: Halacaridae) from Goa, India

Tapas Chatterjee,1 Mandar Nanajkar2

1 Crescent International School, Bario, Govindpur, Dhanbad 828109, Jharkhand, India 2 CSIR, National Institute of Oceanography, Dona Paula, Goa-403004, India

Corresponding author, e-mails: [email protected], [email protected]

Keywords Rhombognathus, halacarid mite, Goa Abstract Rhombognathus scutulatus Bartsch is reported here from Goa, India. World distribution of this species is also provided.

Pierwsze stwierdzenie Rhombognathus scutulatus (Acari: Halacaridae) na Goa, Indie Słowa kluczowe: Rhombognathus, wodopójki z grupy Halacaride, Goa Streszczenie Artykuł prezentuje pierwsze stwierdzenie na Goa (zachodnie wybrzeże Indii) Rhombognathus scutulatus na tle rozmieszczenia gatunku na świecie.

Introduction

Halacarid mites of west coast of India were studied by the first author from Kerala (Chatterjee, Sarma, 1993; Chatterjee, 1995, 2000; Bartsch, Chatterjee, 2001), Maharastra (Chatterjee, Chang, 2004) and Goa (Sarma, Chatterjee, 1993; Chatterjee, 2015, 2018; Chatterjee, Guru, 2011a, b; Chatterjee, Marshall, Guru, Ingole, Pesic, 2012; Chatterjee, Guru, Sorensen, 2013). In the present communication we report Rhombognathus scutulatus Bartsch, 1983 from rocky shore of Goa. Rhombognathus scutulatus was first described from Philippines (Bartsch, 1983). In India, this species was recorded earlier from Andhra Pradesh, Kanya Kumari, Kerala and Andaman & Nicobar Islands (Chatterjee, 1995).

Material and Methods

The material examined for the present study was collected from sediments of the intertidal seaweeds viz. Sargassum, Ulva and Gracillaria from Anjuna beach, Goa. After preliminary observation, one specimen was processed for SEM study.

127 Tapas Chatterjee, Mandar Nanajkar

Study area: Anjuna beach (Lat. 15° 34' 58'' N; Long. 73° 44' 28.54'' E) is located on the northern stretches of Goa state on the West Coast of India. The coast is mainly sandy and rocky shore. The rocky shore has small intertidal rock pools which are rich with diverse marine flora and fauna. The seaweed cover was seen on rocky shore and was more on seaward side represented by species of Sargassum, Hypnea, Spatoglossum, Chaetomorpha, Sphacealria, Cladophora, Dictyota, Gracillaria, Porphyra and Amphiroa. Some of the seaweeds like Sargassum sp. were found submerged in the water mainly in the splash zone area. The calcified genera include Amphiroa sp. & Cheliosporum sp. were found in abundance in the rook pool area. Whereas the rocky areas display large flankof Sargassum in rock pools & crevices. Three common seaweeds Sargassum, Ulva and Gracillaria were collected for the study of mites. The following abbreviations are used in the text, table and figure legends: AE, anterior epimeral plate; PE, posterior epimeral plate; PGS, perigenital setae; SGS, subgenital setae.

Result and Discussion

Rhombognathus scutulatus Bartsch 1983 Rhombognathus scutulatus Bartsch (1983, pp. 413–415, figs. 46–57; 1993, pp. 20–21, figs. 1A–C; 1999, pp. 354–355, figs. 12F–H; 2000, p. 190; 2003, pp. 273–275, figs. 9A–D; 2006, pp. 42–43; 2009, pp. 35–36); Chatterjee (1995, pp. 284–285, figs. 15–19) Chatterjee and De Troch (2000, pp. 187–188, fig. 6); Smit (2011, p. 344); Abé and Etemadi (2014, pp. 15, 16, figs 23, 24). Material Examined: Males and females from Goa – Anjuna beach among rocky algae – Sargassum, Ulva and Gracillaria. Brief Description: All dorsal plates fused in single shield and sculptured with foveae (Figure 1A). Posterodorsal area with a pair of setae. All ventral plates fused to a ventral shield (Figure 1B). AE area with 3 pairs of ventral setae plus one pair of adjunctive marginal setae. Each PE area with 3 ventral, one dorsal plus 1 adjunctive seta. Male with 11–12 pairs plumose PGS and 2 pairs SGS (Figure 1C). Female with 5 pairs PGS and 2 pairs SGS. Gnathostoma small and compact. Palp 4-segmented. Palpal patella (P2) and trochanter without any setae. Palpal telofemur with 1 seta and tibiotarsus with 3 setae. Telofemora I and II with 6 setae (2 ventral and 4 dorsal); telofemora III and IV devoid of any ventral seta and bearing 3 dorsal setae. Tibia I with 5 setae of which 2 ventral pectinate. Legs with carpite on tarsi and devoid of median claw. Lateral claw endoplanate with broad accessory process bearing about 13–15 teeth (Figure 1D). Distribution: This species is widely distributed in the south-western Pacific Ocean and Indian Ocean: Philippines, Singapore, Australia, Sri Lanka, India, Iran, Kenya, Mauritius and New Gunia. Table 1 and Figure 2 show the distribution of this species. Remarks: There are six species of Rhombognathus viz. R. aspidotus Bartsch (2006), R. con- junctus Bartsch (1986), R. parvulus Viets (1939), R. peltatus Viets (1939), R. scutulatus Bartsch and R. similis Bartsch (1977) in which dorsal plates fused in single shield. R. similis was reported from Andaman & Nicobar islands (Chatterjee, 1995) as R. similis may belong to other species and should be considered at present as Rhombognathus sp. Among Rhombognathus species in which dorsal plates fused in single shield, R scutulatus and Rhombognathus sp. have been recorded from Indian Ocean.

128 Report of Rhombognathus scutulatus (Acari: Halacaridae) from Goa, India

Figure 1. Rhombognathus scutulatus Bartsch, SEM figs. A. Dorsal view; B. Ventral view; C. Genital area of male showing PGS and SGS; D. Claw of tarsus III

Table 1. Rhombognathus scutulatus Bartsch: Localities with habitats

Locality Habitat Ocean Reference

1 2 3 4 PTW: Pacific Ocean, Philippines: Negros Island 0–3 m Bartsch (1983) tropical west Australia: Rottnest Is – Bickley point, Nancy cove, ISE: Indian Ocean, Amphibolis sp, Caulerpa sp. Bartsch (1993) Little Armstrong Bay, south east western Australia Australia: Rottnest Is – Algae: Amphiroa sp. ISE: Indian Ocean, Cape Vlamingh, Fish Hook Bartsch (1993) Cystophora sp, Zonaria sp. south east Bay western Australia

129 Tapas Chatterjee, Mandar Nanajkar

1 2 3 4 India: Palm beach, ITE: Indian Ocean, Visakhapatnam, Andhra Among rocky algae Chatterjee (1995) tropical east Pradesh India: Kanya Kumari ITE: Indian Ocean, (=Cape comorin), Tamil Among rocky algae Chatterjee (1995) tropical east Nadu ITE: Indian Ocean, India: Kovalam, Kerala Among rocky algae Chatterjee (1995) tropical east India: Corvin cove, ITE: Indian Ocean, Among coralline algae Chatterjee (1995) Andaman & Nicobar Islands tropical east Australia: Rottnest Is – Sea grass Amphibolis and various ISE: Indian Ocean, Bickley Bay, Bickley point; Bartsch (1999) algae south east Nancy cove Australia: Great Barrier PTW: Pacific Ocean, Reef, Cape Fergusen, AIMS Algae at low tide mark Bartsch (2000) tropical west beach Australia: Great Barrier PTW: Pacific Ocean, Reef, Magnetic Island, Rocky littoral algae Bartsch (2000) tropical west Alma Bay Among sea grass: Thalassia Chatterjee hemprichii, Halophila ovalis, ITW: Indian Ocean, Kenya: Gazi Bay & De Troch H. stipulacea, Halodule wrightii, tropical west (2000) Syringodium isoetifolium Australia: Dampier, Padina sp. (Phaeophyta), ITE: Indian Ocean, Bartsch (2003) northwestern Australia low water edge tropical east Australia: 40 Mile Beach ITE: Indian Ocean, brown algae Padina and Sargassum Bartsch (2003) north of Cape Preston tropical east Australia: East coast ITE: Indian Ocean, of the Burrup turf of small red algae Bartsch (2003) tropical east Peninsula, Watering Cove Singapore: Strait PTW: Pacific Ocean, Small brown and green rocky algae Bartsch (2006) of Singapore, Labrador park tropical west Among Halimeda from a moderately ITE: Indian Ocean, Srilanka: Ahangama Bartsch (2006) exposed fringing reef flat tropical east Mauritius: South of Port ITW: Indian Ocean, Intertidal wave exposed rocky shore Bartsch (2009) Louis, Flic en Flac tropical west New Gunia: Base G beach, PTW: Pacific Ocean, Marine littoral Smit (2011) Jayapura tropical west Iran: Chabahar Beach, ITW: Indian Ocean, Abe & Etemadi Darya Bozorg, Sargassum sp at 1m depth tropical west (2014) Gulf of Oman ITE: Indian Ocean, India: Goa Various rocky algae Present report tropical east

130 Report of Rhombognathus scutulatus (Acari: Halacaridae) from Goa, India

Figure 2. Distribution map of Rhombognathus scutulatus Bartsch

Chatterjee (1996) reported ciliate infestation on R. Scutulatus from Kovalam beach, Kerala. Bartsch (2009) has given a comparative view on the variation in number of tines on the accessory process of lateral claws and length of idiosoma. This species exhibits wide variation in number of tines on the accessory process of lateral claws ranging from 10 to 28 (Bartsch, 2009; Abé, Etemadi, 2014).

Acknowledgement Thanks are due to Dr. Martin V. Sorensen, Natural History Museum of Denmark, University of Copenhagen, Denmark for making the SEM photographs.

References

Abé, H., Etemadi, I. (2014). Two rhombognathine mites (Acari: Halacaridae) from the Gulf of Oman, Iran. Persian Journal of Acarology, 3 (2), 107–119. Bartsch, I. (2006). A new species and new record of Rhombognathus from Singapore (Acari: Halacaridae). Zootaxa, 1120, 41–49. Bartsch, I. (1999). Halacaridae (Acari) from Rottnest Island, Western Australia. Mites on fronds of the seagrass Amphibolis. In: D.I. Walker, F.E. Wells (eds.), The Seagrass Flora and Fauna of Rottnest Island, Western Australia (pp. 333–357). Perth: Western Australian Museum. Bartsch, I. (2000). Rhombognathinae (Acari: Halacaridae) from the Great Barrier Reef, Australia. Memoirs of the Queensland Museum, 45, 165–203. Bartsch, I. (2003). Rhombognathine mites (Halacaridae: Acari) from Dampier, Western Australia: taxonomy and biogeography. In: F.E. Wells, D.I. Walker, D.S. Jones (eds.), The Marine Flora and Fauna of Dampier, Western Australia (pp. 255–280). Perth: Western Australian Museum. Bartsch, I. (1993). Rhombognathine mites (Halacaridae, Acari) from Rottnest Island, Western Australia. In: F.E. Wells, D.I. Walker, D.S. Jones, H. Kirkman, R. Lethbridge (eds.), The Marine Flora and Fauna of Rottnest Island, Western Australia (pp. 19–43). Perth: Western Australian Museum.

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Cite as: Chatterjee, T., Nanajkar, M. (2019). Report of Rhombognathus scutulatus (Acari: Halacaridae) from Goa, India. Acta Biologica, 26, 127–132. DOI: 10.18276/ab.2019.26-11. #1#

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