A high‐throughput method to quantify feeding rates in aquatic organisms: A case study with Daphnia

1. Food ingestion is one of the most basic features of all organisms. However, obtaining precise—and high‐throughput—estimates of feeding rates remains challenging, particularly for small, aquatic herbivores such as zooplankton, snails, and tadpoles. These animals typically consume low volumes of food that are time‐consuming to accurately measure.
2. We extend a standard high‐throughput fluorometry technique, which uses a microplate reader and 96‐well plates, as a practical tool for studies in ecology, evolution, and disease biology. We outline technical and methodological details to optimize quantification of individual feeding rates, improve accuracy, and minimize sampling error.
3. This high‐throughput assay offers several advantages over previous methods, including i) substantially reduced time allotments per sample to facilitate larger, more efficient experiments; ii) technical replicates; and iii) conversion of in vivo measurements to units (mL^‐1 hr^‐1 ind^‐1) which enables broad‐scale comparisons across an array of taxa and studies.
4. To evaluate the accuracy and feasibility of our approach, we use the zooplankton, Daphnia dentifera, as a case study. Our results indicate that this procedure accurately quantifies feeding rates and highlights differences among seven genotypes.
5. The method detailed here has broad applicability to a diverse array of aquatic taxa, their resources, environmental contaminants (e.g., plastics), and infectious agents. We discuss simple extensions to quantify epidemiologically relevant traits, such as pathogen exposure and transmission rates, for infectious agents with oral or trophic transmission.

     

Orientation and ingestion rates of larval Anopheles albimanus in response to floating particles

Response of fourth-instar larvae of Anopheles albimanus Wiedemann (Diptera: Culicidae) to food and inert particles floating at the water surface was studied. In a choice test, larvae aggregated at powdered organic materials (blood meal, liver powder alfalfa flour and wheat flour) but not at inert materials (kaolin, chalk or charcoal). Larvae responded positively to proteins as well as some carbohydrates, but not to cellulose. Retention of larvae at food sources found by random locomotion was found to be responsible for larval aggregation. Larvae ingested food particles 6–9 times faster than insert particles. The significance of Anopheline feeding behavior in the development of formulations of stomach toxins (bacterial agents) used in larval control is discussed.

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Zusammenfassung Die vorliegende Studie befasst sich mit Verhaltensreaktionen von Anopheles albimanus Viertlarven auf an der Wasseroberfläche schwimmende Partikel. Verteilung und Orientierung der Larven wurde in einer Wahlapparatur quantifiziert. Nach Auftrag von Alfalfamehl, Weizenmehl, Stärke, Blutmehl, Leberpulver und Fischmehl wurde Aggregation der Larven in den beköderten Fächern der Apparatur beobachtet. Sowohl Proteine (Casein) als auch einige Kohlehydrate (Amylose, Amylopectin) lösten Aggregationen der Larven aus. Im Unterschied dazu führte Auftrag von Kreide, Kaolin, Polyaethylenpulver, Talcum oder Cellulose nicht zu Aggregationen. Zur Beschreibung der Entstehung larvaler Aggregationen bei Futterstoffen wurden die Schwimmbewegungen der Larven in Anwesenheit von Weizenmehl als Ködersubstanz quantifiziert. Da keine Attraktion der Larven im Sinne einer gerichteten Schwimmbewegung beobachtet werden konnte, wird geschlussfolgert, dass sofortige Beendung der Suchaktivität der Larven bei zufällig gefundenen Futterquellen für die beobachteten Aggregationen bei organischen Substanzen verantwortlich ist.Die Fressraten der Larven bei Angebot verschiedener Substanzen im Überschuss wurde bestimmt. Larven fülten drei von insgesamt sechs Darmabschnitten innerhalb von 15–30 min bei Angebot von Futtersubstanzen, während die Füllung von nur zwei Darmabschnitten mit inerten Materialien erst nach 90–120 min zu beobachten war. Die Resultate werden in Bezug auf wasseroberflächengebundene Formulierungen von Frassgiften diskutiert. Inerte Trägersubstanzen werden wahrscheinlich wesentlich langsamer aufgenommen als Futtersubstanzen. Da An. albimanus Larven nicht von Futterquellen angezogen werden, ist eine rasche und wirksame Giftaufnahme besonders dann zu erwarten, wenn die gesamte Oberfläche der Brutgewässer mit toxinhaltigen Trägerpartikeln bedeckt werden kann.

     

The use of a rapid radiolabeling method for measuring ingestion rates of detritivores

Utilization of the organic components of senescent Spartina alterniflora Loisel detritus by the grass shrimp, Palaemonetes pugio Holthuis, was investigated in laboratory feeding studies. A ¹⁴C-dimethyl sulfate labeling procedure was utilized to label the sterilized detritus. The formed covalent bond is stable to autoclaving, freezing and extremes of pH. An ingestion rate of 2.0 × 10^?4CS·h^?1 was determined for P. pugio. Estimates for rate of incorporation, gross growth efficiency, and total biomass incorporated are presented. An inverse relationship was demonstrated between shrimp size and rates of ingestion and incorporation.     

Functional bioassays utilizing zooplankton: a comparison

Functional zooplankton bioassays based on ingestion, reproduction and respiration are described, with methods for a new ingestion bioassay included. All bioassays are compared using three indices, including the variability of controls, the range of experimental responses, and a listing of contaminants causing inhibition/stimulation of response. The ingestion bioassay showed the greatest range of response, and was sensitive to pesticides, PCBs and heavy metals. It was also commonly characterized by a hormesis response. The reproduction bioassay showed the lowest variability, illustrated a reduced range of response, and was sensitive to nutrients and heavy metals. In one study, the respiration bioassay was sensitive only to PCBs.     

Filtration rates of mosquito larvae in suspensions of latex microspheres and yeast cells

Filtration rates of fourth instars of Aedes aegypti L., Anopheles albimanus Wiedemann, Anopheles quadrimaculatus Say and Culex quinquefasciatus Say (Diptera: Culicidae) were determined by quantifying removal rates of suspended latex microspheres or yeast cells. Average filtration rates were 33–34 l/larva/h (An. quadrimaculatus), 49–55 (An. albimanus), 490–590 (C. quinquefasciatus) or 590–690 l/larva/h (Ae. aegypti) for larvae exposed to latex beads suspended in phagostimulant yeast extract solutions. In suspensions of yeast cells, filtration rates of Ae. aegypti and C. quinquefasciatus were not significantly different from filtration rates in latex bead suspensions. Larval density, ranging from 0.3 to 2.4 individuals/ml in tests with Ae. aegypti and C. quinquefasciatus and up to 4.8 larvae/ml in tests with Anopheles, did not influence filtration rates.

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Zusammenfassung Die Filtrierraten von Viertlarven der Stechmückenarten Aedes aegypti, Anopheles albimanus, Anopheles quadrimaculatus und Culex quinquefasciatus wurden in Laborversuchen bestimmt. Dabei wurde die Filtrierrate definiert als ein Wasservolumen, welches pro Stunde von einer Larve von den Testpartikeln (Hefezellen oder Latexkugeln mit einem Durchmesser von 2 m) befreit wurde. Nach Exposition der Larven in Dichten zwischen 0.15 und 2.4 Larven/ml (Ae. aegypti und C. quinquefasciatus), oder zwischen 0.6 und 4.8 Larven/ml (Anopheles) wurde der Partikelgehalt der Suspensionen in einem elektronischen Partikelzähler bestimmt. Die Filtrierraten wurden über die Verringerung der Partikeldichte mit zunehmender Larvendichte entsprechend einer in der Literatur angegebenen Formel berechnet.Suspensionen von Hefezellen wurden von Ae. aegypti Larven mit einer Leistung von 680±220 l/Larve/h gefiltert. Bei Larven von C. quinquefasciatus wurden Filtrierraten von 600±120 l/Larve/h gemessen. Die Filtrierraten beider Arten waren unabhängig von der Larvendichte. In Suspensionen von Latexpartikeln (zur Phagostimulation der Larven wurden diese Partikel in Lösungen von Hefeextrakt angeboten) wurden die folgenden Filtrierraten festgestellt: An quadrimaculatus: 33–34, An. albimanus: 49–55, C. quinquefasciatus: 490–590, und Ae. aegypti: 560–690 l/Larve/h. Die Larvendichte hatte auch hier keinen Einfluss auf die Filtrierrate. Hefezellen und Latexpartikel wurden von Ae. aegypti und C. quinquefasciatus mit statistisch nicht signifikant verschiedenen Filtrierraten aufgenommen. Die Filtrierraten der Anopheles Larven waren um mehr als eine Zehnerpotenz kleiner als die Filtrierraten von Culex und Aedes, und auch untereinander signifikant verschieden. Der Einfluss der Filtrierraktivität von Stechmückenlarven auf das Seston der Brutgewässer wird diskutiert.

     

Mineral licks as a sodium source for Isle Royale moose

Summary Natural mineral licks and their use by moose (Alces alces) on Isle Royale National Park, Michigan, were studied during 1982–85. The distribution of known licks suggested that they occurred in association with glacial debris, primarily in the western portions of the island. Moose utilized mineral springs extensively during the spring-summer period, and at least 5 licks were used year-round. During summer, a pronounced diel pattern of moose visitation was apparent, with peak use occurring between 0400–0800 h. Although daytime lick use declined by late June, morning and evening use continued to be relatively high throughout the study period. Peak lick use coincided with leaf-emergence in spring. Moose continued to utilize mineral licks despite the availability of ponds containing aquatic plants. Sodium appeared to be the element attracting moose to licks where they ingest copious amounts of water. Observed sodium ingestion rates (0.35 g/min) at licks indicate that licks provide a more concentrated source of sodium compared to aquatic plants (0.023 g/min). Based on the data presented, we reject the conclusions of earlier workers that aquatic plants constitute the only significant source of sodium for Isle Royale moose.     

Genetic and physiological parameters associated with cadmium toxicity in Drosophila melanogaster

Two strains of Drosophila melanogaster represent the extremes in resistance and sensitivity to the lethal effects of CdCl₂. The strain containing the mutations vermilion and brown (v; bw) and the strain Austin had LC₅₀'s of 3.3 and 1.3mm CdCl₂, respectively. The three major chromosomes from these two strains were assorted genetically into the six possible combinations. The measured LC₅₀'s for CdCl₂ for these six genotypes fell into two groups according to the X chromosome; those containing the X chromosome from v; bw had LC₅₀'s 0.5–1.0mm greater than those in which the X chromosome was from Austin. Since the parent strains differed by 2mm, we suggest that the X chromosome is a major, but not the sole, site of genes to produce resistance to CdCl₂. When ¹⁰⁹Cd was in the diet the uptake by v; bw and Austin over 2 days was the same. After 4 days of uptake, the Austin strain excreted the ¹⁰⁹Cd five times faster than v; bw but the six genotypes did not differ appreciably in excretion rate from one another and resembled the sensitive parent Austin more than the resistant one. Thus a second process is indicated that distinguishes resistance to CdCl₂ that apparently is not associated with the X chromosome.This research was sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-84OR21400 with Martin Marietta Energy Systems, Inc.     

Adaptive changes in lipid composition of rat liver plasma membrane during postnatal development following maternal ethanol ingestion

The fatty acid composition of constituent phospholipids and the cholesterol content of rat liver plasma membranes were determined subsequent to maternal alcohol ingestion during pregnancy and lactation. The alcoholic group was given a liquid Metrecal diet containing 37% ethanol-derived calories. The control group was pair-fed an isocaloric sucrose/Metrecal diet. Litters were killed for lipid analyses at days 5, 15 and 25 after birth. These studies revealed that the total phospholipid phosphorus was similar and increased significantly with age in both groups. Cholesterol also increased significantly with age in both groups but was greater in the alcoholic pups, resulting in a higher cholesterol/phospholipid molar ratio. While the phosphatidylethanolamine (PE) content increased with age in both groups, that of sphingomyelin decreased. Phosphatidylserine + phosphatidylinositol (PS + PI) was significantly higher in the control group at all ages studied. A consistent increase of C22:6 in phosphatidylcholine (PC), sphingomyelin, PS + PI and in the total phospholipid fraction from alcoholic pups was observed. Although other fatty acid changes were found in PC, PS + PI and sphingomyelin, PE was not affected. These results suggest that specific adaptive changes were induced in the liver plasma membrane lipids of the progeny from alcoholic rats.     

Experimental Histomoniasis in the Guinea Fowl,Numida meleagris*

Guinea fowl were inoculated rectally with Histomonas meleagridis to produce histomoniasis. The birds were infected readily by this unnatural route. Severe cecal involvement was frequent and long-lasting but liver lesions and death were rare. Turkeys given the same inoculum had high levels of liver involvement and mortality. Guinea fowl responded much less severely to infection with H. meleagridis when infected by a natural route (ingestion of a vector) rather than by rectal inoculation. Thus, naturally acquired infections with H. meleagridis appear to be of small consequence in the guinea fowl.     

Distribution and hydraulic significance of large woody debris in a lowland Australian river

The line-intersect technique was used to measure the loading of large woody debris in a 1.8 km reach of the Thomson River, Victoria (catchment area of 3540 km²). A debris census (measuring every item present) was done over 0.775 km of this reach. The transect technique over-estimated the actual loading revealed by the census. The loading of debris 0.01 m in diameter for the total 1.8 km reach was 0.0172 m³ m^–2, which is higher than that measured in many headwater streams in other parts of the world. The volume loading of debris measured from low level aerial photographs was only 4.8% of the value estimated by the line-intersect technique. The line-intersect estimates were biased due to non-random orientation of debris in the stream (causing estimated errors of +8% for volume loading and +16% for surface area loading). It is recommended that to avoid this problem, when using the line-intersect transect technique in lowland rivers, each line should comprise at least two obliquely-angled transects across the channel. The mean item of debris (0.1 m in diameter) had a trunk basal diameter of 0.45 m, a length of 7.4 m, and volume of 0.7 m³. The riparian trees and the in-channel debris were of similar dimensions. The debris tended to be close to the bed and banks and was oriented downstream by the flow at a median angle of 27°. Because of this orientation, most debris had a small projected cross-sectional area, with the median value being only 1 m². Thus, the blockage ratio (proportion of projected area of debris to channel cross-sectional area) was also low, ranging from 0.0002 to 0.1, with a median value of 0.004. The average item of debris, which occupied only 0.4% of the cross-section, would have minimal influence on banktop flow hydraulics, but the largest items, which occupied around 10%, could be significant. Judicious re-introduction of debris into previously cleared rivers is unlikely to result in a large loss of conveyance, or a detectable increase in flooding frequency.