Integrating host immune status, Labyrinthula spp. load and environmental stress in a seagrass pathosystem: Assessing immune markers and scope of a new qPCR primer set.

Recent trends suggest that marine disease outbreaks caused by opportunistic pathogens are increasing in frequency and severity. One such malady is seagrass wasting disease, caused by pathogens in the genus Labyrinthula. It is suspected that pathogenicity is intimately linked to the ability of the host to initiate defense responses; however, supportive evidence is lacking. To address this, we developed two techniques, including 1) a new qPCR-based pathogen detection method, and 2) an immune profiling panel via four host-biomarker assays (measuring peroxidase, exochitinase, polyphenol oxidase, and lysozyme activities). These techniques were then used to experimentally investigate the impact of environmental stressors (namely, elevated temperature and salinity) on host immunity and how immune status might affect susceptibility to Labyrinthula infection. In the first experiment, we subjected individual turtlegrass (Thalassia testudinum) shoots to short-term (7 d) abiotic stressors alone. In a second experiment, the same abiotic stressor conditions were followed by pathogen exposure (7 additional d), simulating a scenario where we attempt to isolate the impact of environmental stressors on the host seagrass species by removing the stressor as the pathogen is introduced. The qPCR assay successfully quantified the abundance of Labyrinthula spp. cells from both pure cultures and seagrass tissues across a broad range of predominately pathogenic strains, with high sensitivity. Immune enzyme assays revealed that all four biomarkers were constitutively active in turtlegrass individuals, but specific activities were largely unaffected by the chosen abiotic stressor conditions. We also identified positive correlations between pathogen load and two biomarkers (peroxidase, exochitinase), regardless of abiotic stress treatment, further demonstrating the potential utility of these biomarkers in future applications.

Enigmatic Phytomyxid Parasite of the Alien Seagrass Halophila stipulacea: New Insights into Its Ecology, Phylogeny, and Distribution in the Mediterranean Sea.

Marine phytomyxids represent often overlooked obligate biotrophic parasites colonizing diatoms, brown algae, and seagrasses. An illustrative example of their enigmatic nature is the phytomyxid infecting the seagrass Halophila stipulacea (a well-known Lessepsian migrant from the Indo-Pacific to the Mediterranean Sea). In the Mediterranean, the occurrence of this phytomyxid was first described in 1995 in the Strait of Messina (southern Italy) and the second time in 2017 in the Aegean coast of Turkey. Here we investigated, using scuba diving, stereomicroscopy, light and scanning electron microscopy, and molecular methods, whether the symbiosis is still present in southern Italy, its distribution in this region and its relation to the previous reports. From the total of 16 localities investigated, the symbiosis has only been found at one site. A seasonal pattern was observed with exceptionally high abundance (> 40% of the leaf petioles colonized) in September 2017, absence of the symbiosis in May/June 2018, and then again high infection rates (~ 30%) in September 2018. In terms of anatomy and morphology as well as resting spore dimensions and arrangement, the symbiosis seems to be identical to the preceding observations in the Mediterranean. According to the phylogenetic analyses of the 18S rRNA gene, the phytomyxid represents the first characterized member of the environmental clade "TAGIRI-5". Our results provide new clues about its on-site ecology (incl. possible dispersal mechanisms), hint that it is rare but established in the Mediterranean, and encourage further research into its distribution, ecophysiology, and taxonomy.

Temperature-dependent development, cold tolerance, and potential distribution of Cricotopus lebetis (Diptera: Chironomidae), a tip miner of Hydrilla verticillata (Hydrocharitaceae).

A chironomid midge, Cricotopus lebetis (Sublette) (Diptera: Chironomidae), was discovered attacking the apical meristems of Hydrilla verticillata (L.f. Royle) in Crystal River, Citrus Co., Florida in 1992. The larvae mine the stems of H. verticillata and cause basal branching and stunting of the plant. Temperature-dependent development, cold tolerance, and the potential distribution of the midge were investigated. The results of the temperature-dependent development study showed that optimal temperatures for larval development were between 20 and 30°C, and these data were used to construct a map of the potential number of generations per year of C. lebetis in Florida. Data from the cold tolerance study, in conjunction with historical weather data, were used to generate a predicted distribution of C. lebetis in the United States. A distribution was also predicted using an ecological niche modeling approach by characterizing the climate at locations where C. lebetis is known to occur and then finding other locations with similar climate. The distributions predicted using the two modeling approaches were not significantly different and suggested that much of the southeastern United States was climatically suitable for C. lebetis.

Effects of short-term hypersalinity exposure on the susceptibility to wasting disease in the subtropical seagrass Thalassia testudinum.

Seagrass meadows are a vital component of coastal ecosystems and have experienced declines in abundance due to a series of environmental stressors including elevated salinity and incidence of disease. This study evaluated the impacts of short-term hypersalinity stress on the early stages of infection in Thalassia testudinum Banks ex König by assessing changes in cellular physiology and metabolism. Seagrass short shoots were exposed to ambient (30 psu) and elevated (45 psu) salinities for 7 days and subsequently infected for one week by the causative pathogen of wasting disease, Labyrinthula sp. The occurrence of wasting disease was significantly lower in the hypersalinity treatments. Additionally, while exposure to elevated salinity caused a reduction in chlorophyll a and b content, T. testudinum's health, in terms of photochemical efficiency, was not significantly compromised by hypersalinity or infection. In contrast, plant respiratory demand was significantly enhanced as a function of infection. Elevated salinity caused T. testudinum to significantly increase its in vivo H(2)O(2) concentrations to levels that exceeded those which inhibited Labyrinthula growth in a liquid in vitro assay. The results suggest that while short-term exposure to hypersalinity alters selected cellular processes this does not necessarily lead to an immediate increase in wasting disease susceptibility.

Liquid culture and growth quantification of the seagrass pathogen, Labyrinthula spp.

Symptoms characteristic of wasting disease, thought to result from infection by protozoan pathogens (i.e. Labyrinthula spp.), are a common phenomenon affecting seagrass species worldwide. However relatively little is known about factors that control the survival and success of Labyrinthula in part due to the difficulty associated with quantifying the growth of this organism. Here we describe a simple and inexpensive method for measuring growth of Labyrinthula in liquid culture that takes into consideration both cell density and areal spread. The technique allows for examination of the effects of both abiotic and biotic factors on the growth of Labyrinthula apart from its seagrass host, separating the effects of environmental condition on the host from their effects on the pathogen.

Chemical defense in Elodea nuttallii reduces feeding and growth of aquatic herbivorous Lepidoptera.

The submersed macrophyte Elodea nuttallii (Hydrocharitaceae) is invasive in Europe and frequently found in aquatic plant communities. Many invertebrate herbivores, such as larvae of the generalist aquatic moth, Acentria ephemerella (Lepidoptera, Pyralidae), avoid feeding on E. nuttallii and preferably consume native species. First instar larvae exhibited a high mortality on E. nuttallii compared to the native macrophyte Potamogeton perfoliatus. Mortality of older larvae was also high when fed E. nuttallii exposed to high light intensities. Growth of older larvae was strongly reduced on E. nuttallii compared to pondweeds (Potamogeton lucens). Neither differences in nitrogen nor phosphorus content explained the different performance on these submerged macrophytes, but plants differed in their flavonoid content. To investigate whether plant-derived allelochemicals from E. nuttallii affect larval performance in the same way as live plants, we developed a functional bioassay, in which Acentria larvae were reared on artificial diets. We offered larvae Potamogeton leaf disks coated with crude Elodea extracts and partially purified flavonoids. Elodea extracts deterred larvae from feeding on otherwise preferred Potamogeton leaves, and yet, unknown compounds in the extracts reduced growth and survival of Acentria. The flavonoid fraction containing luteolin-7-O-diglucuronide, apigenin-7-O-diglucuronide, and chrysoeriol-7-O-diglucuronide strongly reduced feeding of larvae, but did not increase mortality. The concentrations of these compounds in our assays were 0.01-0.09% of plant dry mass, which is in the lower range of concentrations found in the field (0.02-1.2%). Chemical defense in E. nuttallii thus plays an ecologically relevant role in this aquatic plant-herbivore system.

Hydrilla stems and tubers as hosts for three Bagous species: two introduced biological control agents (Bagous hydrillae and B. affinis) and one native species (B. restrictus).

Field observations suggested that the introduced Hydrilla verticillata (L.f.) Royle biological control agent, a stem weevil, Bagous hydrillae O'Brien, would feed on hydrilla tubers and stems, and a native species, Bagous restrictus LeConte, would feed on hydrilla stems. In choice tests, B. hydrillae readily oviposited in hydrilla tubers. Larval development of B. hydrillae in hydrilla tubers was similar to that in stems; greater adult biomass was attained and less time was needed to complete development when the larvae were fed tubers. Larvae of the hydrilla tuber weevil, B. affinis Hustache, did not complete development in hydrilla stems. Larvae of B. affinis completed development more rapidly when fed new compared with old hydrilla tubers. The native B. restrictus successfully completed development in hydrilla stems, although the larvae required slightly more time compared with the biocontrol agent, B. hydrillae. These findings indicated that feeding on tubers by B. hydrillae may benefit the species particularly when hydrilla stems are seasonably absent or unsuitable especially in more northern climates. In terms of hydrilla control, damage to tubers by this species constitutes a reduction in future infestations of hydrilla propagated by tubers. Finally, hydrilla is suitable to the native weevil, B. restrictus, because larvae completed development in hydrilla stems.

Host plant selection of two Mansonia blanchard species (Diptera: Culicidae) in a heterogeneous habitat of Buenos Aires City, Argentina.

Larvae and pupae of the genus Mansonia Blanchard attach to the roots of aquatic plants by means of modified structures to obtain oxygen. A study of the association of larval Ma. indubitans and Ma. titillans with floating macrophytes was conducted at Macies Pond, Argentina. Fifty-four sampling units were taken from January to May 2003. Three genera of host plants were considered: Pistia, Limnobium, and Salvinia. A total of 402 immatures of Ma. indubitans and 217 of Ma. titillans were captured and associations between Mansonia immatures and roots of each genera were assesed. Significant association was noted between Ma. indubitans and certain host plant species (K-W H=42.74, df=2, p<0.001). The same result was observed for Ma. titillans (K-W H=23.42, df=2, p<0.001). Both Mansonia species utilized roots of P. stratiotes in significantly higher proportions than expected by random selection. Both species showed significant negative association with Salvinia spp., while no clear relationships were detected with L. laevigatum.

Metacystis borrori n. sp. (Ciliophora: Metacystidae) on the seagrass Thalassia testudinum.

A new epibiontic ciliate of the genus Metacystis is described on the seagrass Thalassia testudinum of the coral reef lagoons of Veracruz, Mexico. The ciliate was studied in living and stained specimens and under the scanning electron microscope. The cell body (10-35 x 10-18 microm in vivo) is transversely annulated (4-6 rings). The somatic ciliature consists of 22-30 longitudinal kineties, and patterned as 5-7 transverse kineties. The circumoral kinety is composed of kinetosomes closely spaced. The macronucleus diameter measures about 3-7 microm. The lorica (18-61 x 11-26 microm) has the posterior end round to conical or irregular with mucoid filaments. This prostomatid colonizes both natural and artificial substrates placed in an aquarium. Metacystis borrori n. sp. is a species that forms part of the ciliate community on Thalassia testudinum with a temperature range of 21-26 degrees C and a salinity of 32-40 per thousand.