Contemporary veterinary medicine

Contemporary veterinary medicine

Description and planned scope of the project

Contemporary veterinary medicine continues to grapple pet obesity. Although obesity is commonly observed in human beings, it is becoming common in animals. Pet obesity is described as any bodyweight that is fifteen percent beyond the ideal weight. It is as a result of prolonged imbalances in energy intakes. Nonetheless, the underlying cause of pet obesity is overfeeding. Different researchers have come up with animal models to monitor metabolism and energy levels in animals. The incidence of pet obesity in the USA is on the increase. Nonetheless, the epidemic is widespread across the globe and not restricted in the USA.  Obesity is common in carnivore pets such as cats and dogs. Pet obesity is common in Dachshunds, Basset hounds, Shetland sheepdogs, and Cocker spaniels.  In most cases, managing pet obesity calls for dietary restrictions. Just like people in the West, pets tend to have a sedentary lifestyle that makes them more prone to obesity.

The term pet refers to a companion animal that is primarily kept due to compassion. Common pets are regarded as having relatable personalities, attractive appearances, and high intelligence levels. Cynophiles and ailurophiles are the common types of pets kept across the globe. Other pets may include rodents, hamsters, gerbils, passerines, salamanders, and hermit crabs. Smalls pets are classified as pocket pets while the bovine and equine animals fall under the large pets categories. For the purpose of this study, the term pets will be restricted to both ailurophiles and Cynophiles. They fall under the carnivorous category and are highly susceptible to obesity.  The lack of exercise regimen and healthy feeding patterns make obesity a common health complication in pets. Canine obesity is the most common obesity in animals. There is a close correlation between obesity and shorter lifespans in the affected pets. There is equal risk to secondary health complications.

Goals of the Proposal

The goal of the following proposal to create standardized fielding and exercise procedures that may help counter pet obesity. There will be an investigation of the primary rigger factors of pet obesity and the animals at high-risk levels.  Some of the risk factors that will be considered include genetic factors, predisposed breeds, type of food, owners’ attitudes, medication, as well as endocrine diseases. The proposal is based on the realization that obesity increases the risk of other diseases. Osteoarticular diseases tend to be common among obese animals. Osteoarthritis is also a condition that leads to the degeneration of joints, thus complicating the movements. Musculoskeletal conditions inhibit movement and the quality of life of pets. A common observation in obese animals is that they experience cartilage erosion. The condition reduces the ability to cushion the bones from grinding with each other. Consistent grinding of the bones leads to both pain and inflammation.

Indeed, lean bodies in animals reduce such complications and boost agility. Lean bodies can also aid in the management of the condition.

The goal of this proposal is to develop a standardized, portable kit of field equipment and field/lab protocols that can be used to investigate plankton diversity, abundance, and trophic ecology across the entire MarineGEO network. This project will include investigations of four major components of the plankton community: microbial plankton, phytoplankton, zooplankton, and ichthyoplankton. These four components will give a broad assessment of the plankton community structure and function.

The Zooplankton, Ichthyoplankton, Phytoplankton and Stable Isotope Survey (ZIPSS) kit will be developed at Friday Harbor Labs (FHL) at the University of Washington, and the initial design phase will focus on understanding plankton ecology at eight stations within the Salish Sea. Although FHL has a large oceanographic vessel, ZIPSS will be designed to be deployed in coastal waters using small vessels. This approach is favored because it can facilitate widespread use within the MarineGEO network, as not all current and future MarineGEO partner institutes may have access to large vessels. A focus of the initial design phase at FHL will be to make ZIPSS compact and portable so it can be used at some of the more remote stations within the MarineGEO network. Following an initial development and field-testing phase at FHL (approximately six months), it will be taken to other institutes within the network to test its potential for deployment in different scenarios. Field work at all stations will be completed within the twelve months of the grant, and sample processing will be completed within the first eighteen months. The finalized protocol for all MarineGEO institutes and at least one peer-reviewed publication will be completed at the end of 24 months.

The baseline procedures for the field and lab surveys are discussed below. The equipment and methodologies outlined here are intended to represent the starting point of ZIPSS development. During development, all aspects of this outline will be evaluated for the following metrics:

• Time at each station in the field
• Time processing each sample in the lab
• Portability of the kit
• Cost

Adjustments will be made to the procedures during the timeframe of the grant to maximize the knowledge gained against these factors.

Environmental Parameters

At each station, a suite of environmental parameters will be recorded from surface waters, including temperature, salinity, dissolved oxygen, and nitrate, nitrite, and phosphate concentrations.

Microbial Plankton

Microbial plankton form the base of the planktonic food network and play important roles in nutrient cycling in coastal waters (Gasol and Del Giorgio 2000). Autotrophic bacteria can account for a large portion of primary production in both oligotrophic and nutrient-rich, coastal waters (Giovannoni and Stingl 2005). Altogether, the bacterial community plays a large role in ecosystem metabolism and nutrient cycling. Certain microbial groups, such as the cyanobacteria Prochlorococcus and Synechococcus, are particularly important groups of photosynthetic bacteria and commonly are measured in marine microbial systems (Campbell et al. 1994).

The microbial plankton community will be examined with the use of flow cytometry. Flow cytometry, when used with fluorescent nucleic acid stains, can be used to assess the relative abundance and biomass of microbes in aquatic environments (Gasol and Morán 2015). Flow cytometers use a small volume of water, usually less than one milliliter per sample, to measure the size of particles passing through a laser or other light source. The resolution of flow cytometry varies by instrument, but they can measure the sizes of up to 10,000 cells per second and detect sizes of particles from eukaryotes to viruses (Gasol and Morán 2015). These devices can even distinguish cells that were alive at collection from detritus based on fluorescence.

At each station, a 10 ml water sample will be collected from surface waters and fixed in glutaraldehyde. Samples then will be frozen in a -80°C freezer until analysis on a flow cytometer. Samples will be processed at FHL, although this is a commercially available test, and, after the proposed work is done, field sites would send samples to a central facility for measurement. Particular emphasis will be placed on measuring the abundance of Prochlorococcus and Synechococcus. With this method, the abundance of the microbial plankton community from eukaryotes to viruses, including important cyanobacteria, can be measured.

Phytoplankton

The concentration of chlorophyll-a (chl a) will be measured from surface waters at each station. Two sets of triplicate 1-liter samples will be collected from the field and placed on ice for transport back to the MarineGEO lab. One set immediately will be filtered onto 0.22 µm polycarbonate filters for analysis of total chl a. The second set of water samples will be used for analysis of size-fractionated nanophytoplankton and picophytoplankton chl a. These samples will be prefiltered through 20 µm bolting to remove netphytoplankton (phytoplankton comprised of cell sizes above 20 µm). Then, this sample is filtered again onto 2 µm polycarbonate filters for measurement of nanophytoplankton. This final filtrate will be filtered onto 0.22 µm polycarbonate filters for measurement of picophytoplankton. Chlorophyll-a will be extracted from the filters using 90% acetone and analyzed on a fluorometer. With this method, the relative contributions of three size classes of the phytoplankton community – netphytoplankton, nanophytoplankton, and picophytoplankton – can be calculated. The filtration requires minimal equipment, and the flourometer will be a cell phone based system that can be deployed at any network site. Part of the ZIPSS development process will be the generation of standards for calibrating the cell phone system in the field. The data for this will be compared against a Turner AquaFlash handheld fluorometer.

Zooplankton

Zooplankton will be collected using a 250 µm mesh net towed in surface waters. The mouth of the net will be fitted with a flow meter to estimate the volume of water filtered per tow. The 250 µm mesh size typically is used in zooplankton collections, although it may lead to an underestimate of small copepods (Turner 2004). Five tows will be made at each station. Four of these will be preserved in 100% ethanol for taxonomy while the fifth will be placed on ice for stable isotope analysis.

In the lab, algae and large debris from each of the samples will be removed. Larval fishes from all samples also will be removed for later processing. Each of the four alcohol-preserved samples will be screened through a series of five sieves – 2000 µm, 1000 µm, 500 µm, 180 µm, and 53 µm. One of the four sieved samples will be preserved in 70% ETOH for archiving at the USMN, and the other three will be oven-dried in pre-weighed tins until all moisture is gone. These sieve sizes correspond to particular components of the plankton community (Table 1). This is a faster, cruder approach to zooplankton community assessment than taxonomic identification, but it requires less taxonomic expertise. The filtration method can be completed within a few hours, whereas taxonomy can take up to twelve hours per sample, or even longer if the taxonomist is unfamiliar with the species composition of the samples. This means that a full taxonomic evaluation of even a small sampling effort of 5-10 stations would require between 60-120 hours of lab work. We do not expect every MarineGEO to have a zooplankton taxonomist, or be able to dedicate so much time to taxonomy. At some future time, it may be important to know the exact taxonomic composition of the plankton, and the archival sample will be key to answering questions we do not yet know enough to ask.

Table 1. Zooplankton size fractions and their associated taxonomic groups. From Rolff (2000) and Bănaru et al. (2013).

Ichthyoplankton

Larval fishes extracted from the samples will be identified based on morphology to lowest possible taxon. Each taxon will be enumerated based on the number of individuals and the volume of water filtered per tow.

DNA barcoding – sequencing the 5’ end of the cytochrome c oxidase 1 gene (CO1) – also will be used to identify fish larvae. Barcoding will be used to assess the accuracy of identifications made based on morphology and to provide species-level identifications for small larvae or morphologically indistinguishable genera. At FHL, there will be a particular emphasis on molecular identification of the genus Sebastes, which is one of the most species-rich genera in the Salish Sea with some 27 species present (Pietsch and Orr 2015). While the early life history stages of many Sebastes species are known (Matarese et al. 1989), distinguishing among them remains a challenge due to their overall morphological similarities. Barcoding can distinguish among adult Sebastes (Steinke et al. 2009, Zhang et al. 2013) and should be an effective tool for describing the distribution of Sebastes larvae in the Salish Sea. By combining both a morphological and molecular approach to larval fish taxonomy, the resolution of the larval fish data should be high, allowing for detailed analysis of larval fish distribution within the Salish Sea. At every field site in the MarineGEO network there are similar taxonomic issues to the Sebastes problem of the Salish Sea. This aspect of the tool is being developed in the Salish Sea as a demonstration of the importance and effectiveness of this data collection method.

Stable Isotope Analysis

Stable isotope analysis of carbon (δ¹³C) and nitrogen (δ¹⁵N) will be performed on both zooplankton and ichthyoplankton collections using the frozen samples. Stable isotopes of δ¹³C and δ¹⁵N indicate food web structure and trophic position of organisms, because heavy isotopes are enriched in the food web relative to diet (Michener and Schell 1994). Stable carbon indicates food sources, while stable nitrogen shows trophic level (Hobson and Welch 1992).

Zooplankton samples will be size fractionated from frozen samples as described above, giving at most five size classes of zooplankton. Abundant ichthyoplankton taxa will be measured separately. Tissues will be dried, ground into powder, and placed into tin capsules for analysis. Samples then will be analyzed on a mass spectrometer. This method will allow for the reconstruction of the food web structure of zooplankton and ichthyoplankton at the field sites.

Fit of the research to MarineGEO

Our proposal will directly address multiple core research objectives of MarineGEO. First, we will develop a monitoring system that is simple and versatile to be used at any of the MarineGEO institutes, facilitating the collection and sharing of long-term data using standard protocols. Second, we will assess biodiversity of the plankton community at exemplar sites in the Salish Sea, Gulf of Mexico, and XXXX.  Third, the focus of our proposal is on coastal waters. Finally, we use a multidisciplinary approach to address patterns of biodiversity and ecosystem function.

Contribution to connectivity and collaboration across the MarineGEO network

This research proposal will include direct collaboration among three MarineGEO institutes and has a stated purpose to develop a field sampling kit that will be usable by all current and future MarineGEO partners.

Scientific and broader impacts

Scientific Importance. The ZIPSS system will, for the first time, give us direct insight into the ecological structure of the primary producers and primary consumers in the local food web of the field sites in the MarineGEO network. These data have implications at multiple size and time scales. As site-specific data, they will be useful for understanding the local ecology. As time goes on, the broader spatial data set will capture variability and trends not appreciated in short-term sampling. By comparing sites, which vary in latitude, longitude, and many abiotic factors, we will make more general inferences about the interaction of biotic and abiotic processes. Also, this system is designed to outlast this proposal and spread to all the sites in the MarineGEO network with little investment on the part of each station. That means we will be generating the first data points in what will become a larger, long-term data set that links locality and climate to biology.

Results generated by the ZIPSS system will be presented at local, regional, and international science conferences. At least one manuscript, led by the postdoc, Dr. #1, will be prepared within one year of data collection.

Broader Impacts. The project will develop new infrastructure for the MarineGEO network with the layout and purchasing of the first ZIPSS kits. The training of technical staff in the use of the kits, as well as their deployment will contribute to the knowledgebase of these workers, making them more effective scientists. There has never been a standardized cross-plankton sampling system proposed and we envision that this kit and the protocol will be widely adopted across other field stations and agencies. There has already been interest in this from the National Estuarine Research Reserve System. Lastly, the Co-PI, Dr. #1 has brachial plexus paralysis. This prevents him from full use of his non-dominant hand, and is a visible difference in comparison to other scientists. His whole career, he has been inventing new ways of doing science that make use of his monodexterity. His viewpoint has the potential to change the way sampling systems are designed. He also serves as a visible role model to young people interested in science who might otherwise feel excluded on the basis of some difference from the baseline. His representation as a differently abled scientist will increase the diversity of the scientific endeavor both directly and because he is an inspiration for others.

Proposed role for Postdoc

The largest portion of the budget for this grant is to support the postdoc affiliated with the project, Dr. #1, for two years. #1 has nearly 15 years of experience in managing zooplankton ecology programs and has a specialty in larval fish taxonomy (see Biographical Sketch). Under the supervision of PI #2, #1 will develop the ZIPSS kit and protocols for use across the MarineGEO network. #1 also will lead the field work and lab work for work within the Salish Sea and partner institutes, including organizing and leading field collections, processing samples for DNA and stable isotope analysis, and identifying fish larvae. Professional development will include being lead author on manuscripts, attending conferences to present the work, and giving public talks on the sampling system and the project results.