**5. Integrating species and genetic diversity in one experiment: an alternative approach**

The preceding sections provided an overview of benthic experimental studies that alter either species or genotypic diversity separately. It has been argued that genetic diversity is only important in ecosystems that rely on or are dominated by one or a few key or habitat providing species, such as *Zostera marina* ecosystems (Hughes et al., 2008). However, all living organisms are hierarchically organized: genes make up genotypes, genotypes define populations and populations collectively constitute a species (Reusch & Hughes, 2006). If there is variation and heritability, as well as selection in ecologically important traits such as, e.g., growth rate or resistance to parasites, diversity at any level can have important ecological effects (Hughes et al., 2008). Moreover, it has been shown in terrestrial plant communities that genetic diversity can have direct consequences on species diversity: in a long-term experiment, genetically diverse communities reduced the rate at which species diversity declined (Booth & Grime, 2003), and genetic and species diversity maintained each

Integrating Different Organizational Levels in Benthic

Biodiversity – Ecosystem Functioning (BEF) Studies 103

bacterial community. They established closed microcosms with bacterivore nematode species that colonize decaying organic matter, revealing that intraguild species interactions depend on food availability (Dos Santos et al., 2009). They also worked with semi-closed microcosms of sandy beach sediments with more natural diversity levels (up to 24 species). Results showed that bacterivore nematodes did not have a more pronounced effect on the structure of the bacterial community than nematodes from other trophic guilds; however, nematode species richness (across trophic levels) did (Dos Santos, 2009). Moreover, processes such as carbon production increased with species richness, and the most speciesrich nematode assemblage had a positive effect on bacterial diversity (Dos Santos, 2009).

Fig. 1. The experimental system. a) The experimental unit. Microcosms can be setup in a variety of containers; for the illustration we chose a relatively small glass or plastic jar with a thin, water-saturated layer of sediment, covered by a plastic film or lid to avoid desiccation. Experimental microcosms contain increasing levels of nematode taxonomic and/or genetic diversity b) A schematic view of the benthic food web contained in the sediment of the microcosm. Predatory nematodes prey on herbivorous and bacterivore nematodes, which, in turn, graze on diatoms and prokaryotes respectively. In different experimental units, nematode genetic and species diversity are modified. The effects of the different diversity levels on the functioning of the food web are measured in terms of the production of

To address the BEF subject at different organizational levels simultaneously (i.e., species *and* genetic diversity) across different trophic guilds (i.e., grazers, bacterivores and predators), we follow the microcosm approach as applied in the study by Hubas et al. (2010). We suggest assessing components affecting directly the physical integrity of the habitat as a proxy of the functionality of our microcosms, i.e. the production of EPS and its effect on sediment stability (Figure 1b). However, other functions, such as community respiration, prokaryote enzymatic activities or the decomposition of a specific substrate, may be assessed in the same setup. Microbial EPS are increasingly recognized as a major stabilising factor. They are an ubiquitous component of aquatic ecosystems composed

extracellular polymeric substances (EPS) and sediment stability.

other through mutual feedbacks (Lankau & Strauss, 2007). It is therefore fundamental to start to include changes in genetic (population) and species diversity in studies assessing the consequences of biodiversity decline.

Today, *Zostera marina* vegetations probably provide the best systems to investigate species and genetic diversity in concert (Reusch & Hughes, 2006). To our knowledge, it is also the only system where genotypic (seagrass) and species (grazer) diversity have been manipulated simultaneously (Hughes et al., 2010). One big advantage of this system is that seagrasses can reproduce clonally, which means that multiple shoots of one genotype can be handled as one unit when changing genetic (genotypic) richness just like a species is handled as one unit when changing species richness. This makes it possible to alter species and genotypic richness in one experiment. On the other hand, it is a method restricted to clonally reproducing organisms and cannot be applied to sexually reproducing animals. Another major advantage may be that seagrasses consist of relatively few but functionally diverse species. This is very valuable for the manipulation of the experimental units. However, the results cannot be extrapolated to systems with higher species richness, and questions concerning intraguild diversity may be addressed only to a limited extent. Besides, one major disadvantage of the seagrass system is that a lot of space is needed to set up outdoor (or indoor) mesocosms. Moreover, *Zostera marina* has to be available in order to establish the artificial plantings. Yet, its distribution is limited to the northern hemisphere, mainly along European and North American coasts.

As an alternative approach to test effects of species and genetic diversity on ecosystem functioning, we present a setup allowing experimental manipulation of multitrophic assemblages in very small and easy-to-replicate laboratory microcosms (Figure 1a). These comprise small amounts of sediment containing a benthic microbial food web of diatoms (i.e. primary producers), prokaryotes (i.e. decomposers) and nematodes and/or harpacticoid copepods (i.e. grazers of prokaryotes and of diatoms, as well as predators of other nematodes and copepods) (Figure 1b). These organisms dominate all soft sedimentary habitats such as beaches and mudflats around the world. They typically attain high local diversity, species numbers in 10 ml of sediment typically ranging in the order of hundreds to thousands, and several tens for prokaryotes, diatoms and nematodes, respectively. They are highly relevant to basic benthic ecosystem processes such as the decomposition of organic matter and the mineralization of nutrients. They respond more rapidly to environmental changes than most macrofauna (Bolam et al., 2002).

Former experiments with similar setups have proven to be very useful to study benthic processes. De Mesel et al. (2003, 2004, 2006) studied the impact of bacterivore nematodes on the bacterial community and the decomposition of cordgrass leaves. They found that four different bacterivore nematode species have a significant top-down effect on the structure of the bacterial community (De Mesel et al., 2004). However, species richness within the guild of bacterivore nematodes did not enhance decomposition rates, rather the process depended on species identity and on unexpected inhibitory and facilitative interactions among species within the same guild (De Mesel et al., 2003, 2006). Hubas et al. (2010) investigated the influence of microbial organisms, i.e., bacteria, diatoms and bacterivore nematodes, on the production of extracellular polymeric substances (EPS), a major sediment-stabilizing product (Stal, 2010). The results evidenced that the presence of bacterivore nematodes had a positive impact on microbial abundance and EPS production, which was highest at the highest community complexity, i.e., involving nematodes, diatoms and bacteria (Hubas et al., 2010). Dos Santos (2009) studied bottom-up and top-down controls of nematodes on the

other through mutual feedbacks (Lankau & Strauss, 2007). It is therefore fundamental to start to include changes in genetic (population) and species diversity in studies assessing the

Today, *Zostera marina* vegetations probably provide the best systems to investigate species and genetic diversity in concert (Reusch & Hughes, 2006). To our knowledge, it is also the only system where genotypic (seagrass) and species (grazer) diversity have been manipulated simultaneously (Hughes et al., 2010). One big advantage of this system is that seagrasses can reproduce clonally, which means that multiple shoots of one genotype can be handled as one unit when changing genetic (genotypic) richness just like a species is handled as one unit when changing species richness. This makes it possible to alter species and genotypic richness in one experiment. On the other hand, it is a method restricted to clonally reproducing organisms and cannot be applied to sexually reproducing animals. Another major advantage may be that seagrasses consist of relatively few but functionally diverse species. This is very valuable for the manipulation of the experimental units. However, the results cannot be extrapolated to systems with higher species richness, and questions concerning intraguild diversity may be addressed only to a limited extent. Besides, one major disadvantage of the seagrass system is that a lot of space is needed to set up outdoor (or indoor) mesocosms. Moreover, *Zostera marina* has to be available in order to establish the artificial plantings. Yet, its distribution is limited to the northern hemisphere,

As an alternative approach to test effects of species and genetic diversity on ecosystem functioning, we present a setup allowing experimental manipulation of multitrophic assemblages in very small and easy-to-replicate laboratory microcosms (Figure 1a). These comprise small amounts of sediment containing a benthic microbial food web of diatoms (i.e. primary producers), prokaryotes (i.e. decomposers) and nematodes and/or harpacticoid copepods (i.e. grazers of prokaryotes and of diatoms, as well as predators of other nematodes and copepods) (Figure 1b). These organisms dominate all soft sedimentary habitats such as beaches and mudflats around the world. They typically attain high local diversity, species numbers in 10 ml of sediment typically ranging in the order of hundreds to thousands, and several tens for prokaryotes, diatoms and nematodes, respectively. They are highly relevant to basic benthic ecosystem processes such as the decomposition of organic matter and the mineralization of nutrients. They respond more rapidly to

Former experiments with similar setups have proven to be very useful to study benthic processes. De Mesel et al. (2003, 2004, 2006) studied the impact of bacterivore nematodes on the bacterial community and the decomposition of cordgrass leaves. They found that four different bacterivore nematode species have a significant top-down effect on the structure of the bacterial community (De Mesel et al., 2004). However, species richness within the guild of bacterivore nematodes did not enhance decomposition rates, rather the process depended on species identity and on unexpected inhibitory and facilitative interactions among species within the same guild (De Mesel et al., 2003, 2006). Hubas et al. (2010) investigated the influence of microbial organisms, i.e., bacteria, diatoms and bacterivore nematodes, on the production of extracellular polymeric substances (EPS), a major sediment-stabilizing product (Stal, 2010). The results evidenced that the presence of bacterivore nematodes had a positive impact on microbial abundance and EPS production, which was highest at the highest community complexity, i.e., involving nematodes, diatoms and bacteria (Hubas et al., 2010). Dos Santos (2009) studied bottom-up and top-down controls of nematodes on the

consequences of biodiversity decline.

mainly along European and North American coasts.

environmental changes than most macrofauna (Bolam et al., 2002).

bacterial community. They established closed microcosms with bacterivore nematode species that colonize decaying organic matter, revealing that intraguild species interactions depend on food availability (Dos Santos et al., 2009). They also worked with semi-closed microcosms of sandy beach sediments with more natural diversity levels (up to 24 species). Results showed that bacterivore nematodes did not have a more pronounced effect on the structure of the bacterial community than nematodes from other trophic guilds; however, nematode species richness (across trophic levels) did (Dos Santos, 2009). Moreover, processes such as carbon production increased with species richness, and the most speciesrich nematode assemblage had a positive effect on bacterial diversity (Dos Santos, 2009).

Fig. 1. The experimental system. a) The experimental unit. Microcosms can be setup in a variety of containers; for the illustration we chose a relatively small glass or plastic jar with a thin, water-saturated layer of sediment, covered by a plastic film or lid to avoid desiccation. Experimental microcosms contain increasing levels of nematode taxonomic and/or genetic diversity b) A schematic view of the benthic food web contained in the sediment of the microcosm. Predatory nematodes prey on herbivorous and bacterivore nematodes, which, in turn, graze on diatoms and prokaryotes respectively. In different experimental units, nematode genetic and species diversity are modified. The effects of the different diversity levels on the functioning of the food web are measured in terms of the production of extracellular polymeric substances (EPS) and sediment stability.

To address the BEF subject at different organizational levels simultaneously (i.e., species *and* genetic diversity) across different trophic guilds (i.e., grazers, bacterivores and predators), we follow the microcosm approach as applied in the study by Hubas et al. (2010). We suggest assessing components affecting directly the physical integrity of the habitat as a proxy of the functionality of our microcosms, i.e. the production of EPS and its effect on sediment stability (Figure 1b). However, other functions, such as community respiration, prokaryote enzymatic activities or the decomposition of a specific substrate, may be assessed in the same setup. Microbial EPS are increasingly recognized as a major stabilising factor. They are an ubiquitous component of aquatic ecosystems composed

Integrating Different Organizational Levels in Benthic

Biodiversity – Ecosystem Functioning (BEF) Studies 105

for over 15 years now (T. Moens, unpublished data). Therefore, the premise is that cultured nematodes have low genetic diversity due to inbreeding, unlike natural populations, which are expected to exhibit higher genetic diversity levels. This approach implies that the level of genetic difference has to be assessed before the experiment, which in view of the substantial cryptic diversity (e.g., Derycke et al., 2005) is not necessarily evident. Alternatively, contrasting genetic diversity levels can be obtained experimentally with cultures of selected nematode species. Cultures of species of the *Rhabditis marina* species complex, for instance, can be raised from single gravid females, yielding highly inbred lineages, which can be maintained easily for many generations. It is therefore possible to raise a culture collection of many inbred lineages and to establish experimental populations of very low to high genetic diversity. The advantage of this approach is that similar to the study by Gamfeldt & Kallstrom (2007), "families" can be obtained, and the level of genetic diversity (i.e. the

The main ecosystem function we concentrate on in this approach is measured in terms of EPS production (concentration and composition), and the stability of the sediments. To assess EPS concentration and composition, sediment samples are mixed with distilled water. Exopolymers are very diverse and complex molecules that are found in a wide variety of forms and structures in aquatic ecosystems. The selection of the protocol for their extraction, purification and subsequently for the measurement of their concentration in any sample will depend ultimately on the type of exopolymer studied (e.g., Panagiatopoulos & Sempéré, 2005; Danovaro, 2010). Exopolymers can be found dissolved in the water column or as gels in biofilms and aggregates so that their extraction and quantification is a primary step in assessing their potential availability (Decho, 1990). A description and evaluation of different methods and protocols is well beyond the scope of this paper, hence we will only briefly mention methods that are commonly used in the literature and which offer an acceptable

The water-extractable fraction (i.e. dissolved or colloidal EPS which are already present in pore-water and unbound to sediment grains) can be analyzed for carbohydrates and proteins following the phenolsulphuric acid protocol (DuBois et al., 1956) and the modified Lowry procedure (Raunkjaer et al., 1994). Carbohydrate and protein concentrations can be measured with a UV/VIS spectrophotometer and their concentrations are deduced from

Sediment stability can be estimated through the measurement of the erosion shear stress of the biofilm. Typically, it is possible to measure this using flumes (Jonsson et al., 2006) or *in situ* devices such as the Cohesive Strength Meter (CSM). This device has been used extensively in marine ecosystems since the pioneering work of Paterson (1989) and is an efficient way to estimate sediment erodibility. Mechanical properties of the biofilms in terms of surface adhesion have been developed relatively recently. The method is based on the magnetic attraction of magnetic particles (Magnetic Particle Induction - MagPI; Larson et al., 2009) and is both relatively easy to set up and inexpensive. Specifically, a known volume of ferromagnetic fluorescent particles is spread onto a defined area of the sediment surface. The particles are then recaptured by an overlying electromagnet and the force (magnetic flux) needed to retrieve the particles is determined as a measure of the retentive capacity of the substratum, a proxy for adhesion. This method is suitable for sensitive recording of

calibration curves assessed from glucose and bovine serum albumin, respectively.

"number of families") for each experimental group can be determined *a priori*.

**5.1.2 Measuring EPS production and sediment stability**

ratio of information obtained to amount of time invested.

changes of the surface adhesion of sediments or biofilms.

primarily of carbohydrates and proteins. They have multiple roles and functions: attachment to substrata, flotation and locomotion, feeding, protection against environmental factors such as desiccation, UV radiation and pollution, as well as the development of biofilms (Decho, 1990). Perhaps the most striking feature of these compounds is their ability to bind sediment grains together, enhancing their cohesive strength and thereby increasing resistance to erosion. Particularly diatoms are the major ecosystem engineers of this sediment bio-stabilisation (Stal, 2010), and prokaryotes are increasingly recognised as playing a key role in enhancing sediment surface adhesion and cohesive strength (Gerbersdorf et al., 2008, 2009; Lubarsky et al., 2010). The advantage of this alternative approach is that it offers the possibility of testing both effects of species and genetic diversity on ecosystem functioning.

#### **5.1 Methodology**

#### **5.1.1 Microcosm setup**

To setup the microcosm, a variety of small containers can be suitable, depending on the exact aim of the study. First, a specified amount of non-cohesive sedimentary substrate needs to be placed on the bottom. For example, acid-washed marine sand (Hubas et al., 2010) or non-cohesive glass beads (Lubarsky et al., 2010) can be used. The sediment needs to be fully hydrated by sterile seawater of a specific salinity (which depends on the organisms). Whether or not the seawater needs to cover the sediment depends on the specific aims of the study. Prokaryote and diatom cultures can be established by sampling sediment surface (0 – 5 mm) from natural tidal flats, and re-suspending the sediment slurry in culture media. Alternatively, or additionally, prokaryote and diatom strains can be obtained from established culture collections. Microbial assemblages should then be incubated for several weeks (e.g., Ribalet et al., 2008). The resulting assemblages can then be used for the experiment. An appropriate culture medium, which favours algal rather than bacterial growth, should be applied. F/2 medium is, for instance, widely used for growing marine algae, especially diatoms (Guillard, 1975). If the use of antibiotics cannot be avoided, a proper test of the effect of each antibiotic on the natural assemblage culture should be conducted.

Finally, nematodes can be placed into the microcosms, according to the experimental groups. There are two different approaches in order to obtain different taxonomic (i.e. species richness) diversity levels across trophic groups of nematodes: either from the field or from cultures. For the former, nematode assemblages can be sampled along diversity gradients from, e.g., an intertidal sandy beach (Gingold et al., 2010) or from different beaches (Dos Santos, 2009). They are extracted live through decantation over a 32µm sieve to obtain stock suspensions of different diversity levels. For the latter, different nematode species can be raised under artificial conditions (Moens & Vincx, 1998). Unfortunately, only a limited number of marine nematode species have hitherto been maintained in permanent monospecific cultures, and most of these belong to the same feeding guild, i.e. bacterivores. However, a majority of species from intertidal sediments can be easily extracted from freshly collected sediment and remain active in a variety of laboratory incubation conditions for several weeks (Moens & Vincx, 1998).

In order to establish microcosms containing nematodes of different genetic diversity, lab cultures and field-collected specimens of the same bacteria-feeding species from which permanent cultures exist can be used. Bacteria feeding nematode species such as *Rhabditis marina, Diplolaimelloides meyli, D. oschei,* and *Diplolaimella dievengatensis* have been cultured

primarily of carbohydrates and proteins. They have multiple roles and functions: attachment to substrata, flotation and locomotion, feeding, protection against environmental factors such as desiccation, UV radiation and pollution, as well as the development of biofilms (Decho, 1990). Perhaps the most striking feature of these compounds is their ability to bind sediment grains together, enhancing their cohesive strength and thereby increasing resistance to erosion. Particularly diatoms are the major ecosystem engineers of this sediment bio-stabilisation (Stal, 2010), and prokaryotes are increasingly recognised as playing a key role in enhancing sediment surface adhesion and cohesive strength (Gerbersdorf et al., 2008, 2009; Lubarsky et al., 2010). The advantage of this alternative approach is that it offers the possibility of testing both effects of species

To setup the microcosm, a variety of small containers can be suitable, depending on the exact aim of the study. First, a specified amount of non-cohesive sedimentary substrate needs to be placed on the bottom. For example, acid-washed marine sand (Hubas et al., 2010) or non-cohesive glass beads (Lubarsky et al., 2010) can be used. The sediment needs to be fully hydrated by sterile seawater of a specific salinity (which depends on the organisms). Whether or not the seawater needs to cover the sediment depends on the specific aims of the study. Prokaryote and diatom cultures can be established by sampling sediment surface (0 – 5 mm) from natural tidal flats, and re-suspending the sediment slurry in culture media. Alternatively, or additionally, prokaryote and diatom strains can be obtained from established culture collections. Microbial assemblages should then be incubated for several weeks (e.g., Ribalet et al., 2008). The resulting assemblages can then be used for the experiment. An appropriate culture medium, which favours algal rather than bacterial growth, should be applied. F/2 medium is, for instance, widely used for growing marine algae, especially diatoms (Guillard, 1975). If the use of antibiotics cannot be avoided, a proper test of the effect of each antibiotic on the natural assemblage culture

Finally, nematodes can be placed into the microcosms, according to the experimental groups. There are two different approaches in order to obtain different taxonomic (i.e. species richness) diversity levels across trophic groups of nematodes: either from the field or from cultures. For the former, nematode assemblages can be sampled along diversity gradients from, e.g., an intertidal sandy beach (Gingold et al., 2010) or from different beaches (Dos Santos, 2009). They are extracted live through decantation over a 32µm sieve to obtain stock suspensions of different diversity levels. For the latter, different nematode species can be raised under artificial conditions (Moens & Vincx, 1998). Unfortunately, only a limited number of marine nematode species have hitherto been maintained in permanent monospecific cultures, and most of these belong to the same feeding guild, i.e. bacterivores. However, a majority of species from intertidal sediments can be easily extracted from freshly collected sediment and remain active in a variety of laboratory incubation conditions

In order to establish microcosms containing nematodes of different genetic diversity, lab cultures and field-collected specimens of the same bacteria-feeding species from which permanent cultures exist can be used. Bacteria feeding nematode species such as *Rhabditis marina, Diplolaimelloides meyli, D. oschei,* and *Diplolaimella dievengatensis* have been cultured

and genetic diversity on ecosystem functioning.

**5.1 Methodology 5.1.1 Microcosm setup**

should be conducted.

for several weeks (Moens & Vincx, 1998).

for over 15 years now (T. Moens, unpublished data). Therefore, the premise is that cultured nematodes have low genetic diversity due to inbreeding, unlike natural populations, which are expected to exhibit higher genetic diversity levels. This approach implies that the level of genetic difference has to be assessed before the experiment, which in view of the substantial cryptic diversity (e.g., Derycke et al., 2005) is not necessarily evident. Alternatively, contrasting genetic diversity levels can be obtained experimentally with cultures of selected nematode species. Cultures of species of the *Rhabditis marina* species complex, for instance, can be raised from single gravid females, yielding highly inbred lineages, which can be maintained easily for many generations. It is therefore possible to raise a culture collection of many inbred lineages and to establish experimental populations of very low to high genetic diversity. The advantage of this approach is that similar to the study by Gamfeldt & Kallstrom (2007), "families" can be obtained, and the level of genetic diversity (i.e. the "number of families") for each experimental group can be determined *a priori*.

#### **5.1.2 Measuring EPS production and sediment stability**

The main ecosystem function we concentrate on in this approach is measured in terms of EPS production (concentration and composition), and the stability of the sediments. To assess EPS concentration and composition, sediment samples are mixed with distilled water. Exopolymers are very diverse and complex molecules that are found in a wide variety of forms and structures in aquatic ecosystems. The selection of the protocol for their extraction, purification and subsequently for the measurement of their concentration in any sample will depend ultimately on the type of exopolymer studied (e.g., Panagiatopoulos & Sempéré, 2005; Danovaro, 2010). Exopolymers can be found dissolved in the water column or as gels in biofilms and aggregates so that their extraction and quantification is a primary step in assessing their potential availability (Decho, 1990). A description and evaluation of different methods and protocols is well beyond the scope of this paper, hence we will only briefly mention methods that are commonly used in the literature and which offer an acceptable ratio of information obtained to amount of time invested.

The water-extractable fraction (i.e. dissolved or colloidal EPS which are already present in pore-water and unbound to sediment grains) can be analyzed for carbohydrates and proteins following the phenolsulphuric acid protocol (DuBois et al., 1956) and the modified Lowry procedure (Raunkjaer et al., 1994). Carbohydrate and protein concentrations can be measured with a UV/VIS spectrophotometer and their concentrations are deduced from calibration curves assessed from glucose and bovine serum albumin, respectively.

Sediment stability can be estimated through the measurement of the erosion shear stress of the biofilm. Typically, it is possible to measure this using flumes (Jonsson et al., 2006) or *in situ* devices such as the Cohesive Strength Meter (CSM). This device has been used extensively in marine ecosystems since the pioneering work of Paterson (1989) and is an efficient way to estimate sediment erodibility. Mechanical properties of the biofilms in terms of surface adhesion have been developed relatively recently. The method is based on the magnetic attraction of magnetic particles (Magnetic Particle Induction - MagPI; Larson et al., 2009) and is both relatively easy to set up and inexpensive. Specifically, a known volume of ferromagnetic fluorescent particles is spread onto a defined area of the sediment surface. The particles are then recaptured by an overlying electromagnet and the force (magnetic flux) needed to retrieve the particles is determined as a measure of the retentive capacity of the substratum, a proxy for adhesion. This method is suitable for sensitive recording of changes of the surface adhesion of sediments or biofilms.

Integrating Different Organizational Levels in Benthic

temperature of the environment.

**6. Conclusions**

Biodiversity – Ecosystem Functioning (BEF) Studies 107

experiment would not reflect absolute temporal scales of climate change, it may well reflect and therefore be representative for the rates of temperature change long-lived organisms might experience (Petchey et al., 1999). Knowing that genetic diversity enhanced resistance of seagrass *Zostera marina* to climate extremes (Reusch et al., 2005), it would be particularly interesting to plan hypotheses involving genetic diversity of nematodes and increased

A further advantage of our system is that it includes different trophic levels, from decomposers (prokaryotes) and producers (diatoms) to grazers (bacterivorous and herbivorous nematodes) and their predators (predatory nematodes), some of the latter being omnivorous rather than strictly predacious (Moens et al., 2004). Moreover, for some setups, we can integrate species of a natural community and not only the dominant species of a given habitat. This is important because even weak interactions can have important stabilizing effects on communities (Berlow, 1999). Contrary to the seagrass system, which alters diversity at the primary-producer level, we can assess the effect of species and genetic diversity also at the consumer level. This is important, as it has been suggested that the stronger top-down control in the sea relative to terrestrial habitats (Shurin et al., 2002) implies that in marine systems ecosystem functions such as primary production may be influenced more by herbivores and predators than by plant diversity (Duffy, 2003; Paine, 2002). In order to track and quantify trophic pathways, pulse-chase experiments which label a particular component and trace transfer to consumers can be designed (Middelburg et al., 2000; Moens et al., 2002; Van Oevelen et al., 2006). Such a characterization and quantification of the relationships among the different components of the community would be an additional improvement, since experimental research on multi-level food webs should not only assess the consequences of species richness and identity on ecosystem processes, but also evaluate trophic cascades and the distribution of interaction strengths within natural

communities and how they change with community composition (Duffy, 2002).

after which treatment effects are likely to become confounded by bottling effects.

However, there are probably three major disadvantages that have to be taken in account when planning the experiments: first, only a limited number of species of nematodes can be cultured with the present methods. Second, the identification of nematodes and diatoms needs considerable expertise, especially when working with field samples containing many different species. And third, the design essentially relies on closed microcosms, hence excluding the role of immigration and emigration. Moreover, by bottling aqueous sediments, a system is created which accumulates end products of decomposition and depletes oxygen. As a consequence, the microcosms can only be used for a limited duration,

Our experimental system provides a good alternative to investigate effects of species and genetic diversity in concert. It is easy to setup and contains organisms that are easily accessible. Moreover, it provides the possibility to be extended and address current challenges of BEF experiments. One of these challenges consists in that future BEF studies must go beyond experiments relating some selected species, singly and in combination, to some ecosystem process. Rather, they must address environmental heterogeneity in space or time, which can be captured in long-term studies on one hand, and working with natural communities on the other. Our experimental units as described here are closed systems, however, they can possibly be converted to semi-closed systems and be setup outdoors. Like

#### **5.1.3 Assessing community composition**

We suggest assessing nematode, prokaryote, and diatom community diversity and composition at a genetic and taxonomic level. Most studies identify nematode species morphologically, nematodes first being mounted on permanent slides (Platt & Warwick, 1988). Species are identified with a microscope, using pictorial (Platt & Warwick 1983, 1988; Warwick et al., 1998) taxonomic keys and primary literature on species descriptions, which is becoming increasingly available in online public databases such as NeMys (Deprez et al., 2005). Morphology based identification of diatoms is achieved by fixing the sampling cores in 4% glutaraldehyde and then embedding in Naphrax after chemically removing organic matter using potassium permanganate. The species composition of the microalgal community is assessed by light microscopy using identification keys (Krammer & Lange-Bertalot, 1986-1991; Lange-Bertalot, 1997; Pankow, 1990; Simonsen, 1962; Underwood et al., 1998; Witkowski et al., 2000). However, species diversity can be assessed using a metagenetic approach as well. The three sets of taxa (nematodes, prokaryotes and diatoms) may be pyrosequenced after distinct sampling procedures. Nematodes are extracted from sediment by sieving (45 µm mesh) and floatation techniques and genotyped using both small and large rDNA subunits (Creer et al., 2010; Porazinska et al., 2009, 2010) and/or mitochondrial genes such as part of the cytochrome oxidase subunit I gene (Derycke et al., 2010).  Prokaryotes are assessed from direct DNA extractions of sediment samples and genotyped using 16S rDNA (Coolon et al., 2010; Xu, 2006). When studying well-documented communities, where sequence data is available for a majority of species, such an approach can yield information on species composition, relative abundances and diversity. In the absence of such a reference database (currently unavailable for any marine assemblage, but this will undoubtedly change in the near future), it can still provide an estimate of diversity in terms of both species richness and evenness. Metagenetic analyses yield information on the different genotypes present, and thus allow simultaneous assessment of species as well as intraspecific diversity, pending proper knowledge on barcoding gaps in the taxa of interest.

#### **5.1.4 Advantages/disadvantages of our system**

As a model system for BEF research, our setup combines many advantages. One major advantage from the practical point of view may be that it can be manipulated in small containers and yet contain diversity levels representative of natural communities. The microcosms can easily be incubated under controlled conditions and sufficiently replicated. Nematodes, diatoms and prokaryotes are key players in almost any coastal or stream soft sediment, performing similar essential ecosystem functions across a range of habitats. Therefore they are easily collectable, either at a sandy beach or tidal flat at seashore or from a stream or lake when working with freshwater organisms. A whole experimental setup fits in any average-sized laboratory. If enough space is available, it can even be expanded to bigger mesocosm units including macrofauna as an additional trophic and functional level, which is likely to impact on nematodes, diatoms and prokaryotes through trophic as well as physical interactions (Austen & Widdicombe, 1998; Braeckman et al., 2011). Other extensions are also possible, for example a temperature treatment can be applied to simulate temperature rise as a consequence of climate change. Although the duration of such a short

We suggest assessing nematode, prokaryote, and diatom community diversity and composition at a genetic and taxonomic level. Most studies identify nematode species morphologically, nematodes first being mounted on permanent slides (Platt & Warwick, 1988). Species are identified with a microscope, using pictorial (Platt & Warwick 1983, 1988; Warwick et al., 1998) taxonomic keys and primary literature on species descriptions, which is becoming increasingly available in online public databases such as NeMys (Deprez et al., 2005). Morphology based identification of diatoms is achieved by fixing the sampling cores in 4% glutaraldehyde and then embedding in Naphrax after chemically removing organic matter using potassium permanganate. The species composition of the microalgal community is assessed by light microscopy using identification keys (Krammer & Lange-Bertalot, 1986-1991; Lange-Bertalot, 1997; Pankow, 1990; Simonsen, 1962; Underwood et al., 1998; Witkowski et al., 2000). However, species diversity can be assessed using a metagenetic approach as well. The three sets of taxa (nematodes, prokaryotes and diatoms) may be pyrosequenced after distinct sampling procedures. Nematodes are extracted from sediment by sieving (45 µm mesh) and floatation techniques and genotyped using both small and large rDNA subunits (Creer et al., 2010; Porazinska et al., 2009, 2010) and/or mitochondrial genes such as part of the cytochrome oxidase subunit I gene (Derycke et al., 2010).  Prokaryotes are assessed from direct DNA extractions of sediment samples and genotyped using 16S rDNA (Coolon et al., 2010; Xu, 2006). When studying well-documented communities, where sequence data is available for a majority of species, such an approach can yield information on species composition, relative abundances and diversity. In the absence of such a reference database (currently unavailable for any marine assemblage, but this will undoubtedly change in the near future), it can still provide an estimate of diversity in terms of both species richness and evenness. Metagenetic analyses yield information on the different genotypes present, and thus allow simultaneous assessment of species as well as intraspecific diversity, pending proper

As a model system for BEF research, our setup combines many advantages. One major advantage from the practical point of view may be that it can be manipulated in small containers and yet contain diversity levels representative of natural communities. The microcosms can easily be incubated under controlled conditions and sufficiently replicated. Nematodes, diatoms and prokaryotes are key players in almost any coastal or stream soft sediment, performing similar essential ecosystem functions across a range of habitats. Therefore they are easily collectable, either at a sandy beach or tidal flat at seashore or from a stream or lake when working with freshwater organisms. A whole experimental setup fits in any average-sized laboratory. If enough space is available, it can even be expanded to bigger mesocosm units including macrofauna as an additional trophic and functional level, which is likely to impact on nematodes, diatoms and prokaryotes through trophic as well as physical interactions (Austen & Widdicombe, 1998; Braeckman et al., 2011). Other extensions are also possible, for example a temperature treatment can be applied to simulate temperature rise as a consequence of climate change. Although the duration of such a short

**5.1.3 Assessing community composition**

knowledge on barcoding gaps in the taxa of interest.

**5.1.4 Advantages/disadvantages of our system**

experiment would not reflect absolute temporal scales of climate change, it may well reflect and therefore be representative for the rates of temperature change long-lived organisms might experience (Petchey et al., 1999). Knowing that genetic diversity enhanced resistance of seagrass *Zostera marina* to climate extremes (Reusch et al., 2005), it would be particularly interesting to plan hypotheses involving genetic diversity of nematodes and increased temperature of the environment.

A further advantage of our system is that it includes different trophic levels, from decomposers (prokaryotes) and producers (diatoms) to grazers (bacterivorous and herbivorous nematodes) and their predators (predatory nematodes), some of the latter being omnivorous rather than strictly predacious (Moens et al., 2004). Moreover, for some setups, we can integrate species of a natural community and not only the dominant species of a given habitat. This is important because even weak interactions can have important stabilizing effects on communities (Berlow, 1999). Contrary to the seagrass system, which alters diversity at the primary-producer level, we can assess the effect of species and genetic diversity also at the consumer level. This is important, as it has been suggested that the stronger top-down control in the sea relative to terrestrial habitats (Shurin et al., 2002) implies that in marine systems ecosystem functions such as primary production may be influenced more by herbivores and predators than by plant diversity (Duffy, 2003; Paine, 2002). In order to track and quantify trophic pathways, pulse-chase experiments which label a particular component and trace transfer to consumers can be designed (Middelburg et al., 2000; Moens et al., 2002; Van Oevelen et al., 2006). Such a characterization and quantification of the relationships among the different components of the community would be an additional improvement, since experimental research on multi-level food webs should not only assess the consequences of species richness and identity on ecosystem processes, but also evaluate trophic cascades and the distribution of interaction strengths within natural communities and how they change with community composition (Duffy, 2002).

However, there are probably three major disadvantages that have to be taken in account when planning the experiments: first, only a limited number of species of nematodes can be cultured with the present methods. Second, the identification of nematodes and diatoms needs considerable expertise, especially when working with field samples containing many different species. And third, the design essentially relies on closed microcosms, hence excluding the role of immigration and emigration. Moreover, by bottling aqueous sediments, a system is created which accumulates end products of decomposition and depletes oxygen. As a consequence, the microcosms can only be used for a limited duration, after which treatment effects are likely to become confounded by bottling effects.


**About the Editors**

**Heather Eicher-Miller**, Associate Professor of Nutrition Science, Purdue University has discovered adverse dietary and health outcomes associated with food insecurity and developed novel interventions to improve food security. She also creates new analytical and methodological techniques to quantify and evaluate the relationship between food insecurity, diet, and health.

**Marie Kainoa Fialkowski Revilla**, Associate Professor of Human Nutrition at the University of Hawai'i at Manoa, research focuses on gathering more comprehensive diet and health-related ¯ information on minority and indigenous populations so that more culturally appropriate health promotion activities may be developed.

### *Editorial* **Nutrition among Vulnerable U.S. Populations**

#### **Heather A. Eicher-Miller 1,\* and Marie K. Fialkowski 2**

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +1-765-494-6815

Received: 2 October 2020; Accepted: 12 October 2020; Published: 15 October 2020

**Keywords:** food security; food insecurity; low resource; nutrition; diet; health; food access; food environment; interventions; U.S. population

Food insecurity and low resources continue to be a burden influencing the health, well-being, growth and development of millions of U.S. children and adults [1–4]. Individuals and families experiencing restrained access to food may be concentrated in certain geographic areas or distributed throughout communities. Sometimes groups managing the situation of little or no food resources are even unknown because of their isolated situations. They include all ages, groups of varying races/ethnicities, diverse household compositions, those living in rural and urban areas and many others [1,2]. Many of these groups, both hidden and visible, have rates of food insecurity well above the national average and are influenced by persistent conditions which are historically resistant to trends of national improvement in food security [1,5,6]. Yet, even national food security estimate trends are currently in flux as environmental influences such as the coronavirus pandemic and economic changes shape the food landscape of the U.S. [7]. Research attention to these subsets of the population and varying environmental influences are imperative to determine U.S. health, well-being and nutritional status associated with food insecurity and to use this information to improve these conditions.

Not enough is known about the nutritional status and dietary intake in the diverse array of low-resource and food insecure groups despite summary information regarding the broad group of U.S. children and adults. Some of these subsets may be missed in national surveillance for reasons such as limited samples to make robust estimates, non-response or attrition [8,9]. Nor are the environments and nutritional barriers of the diversity of vulnerable population groups affected by food insecurity and low resources fully understood [10,11]. Creating interventions that effectively intervene to improve food security and nutritional status, however, are dependent on this knowledge as broad, summary information may not translate to a one-size-fits-all approach to improve food security in such a varied food landscape. Tailored approaches to quantify access to food, the nutrition environment, dietary behaviors and other barriers are necessary to identify the needs in diverse populations and then to build successful interventions that will improve dietary intake, reduce rates of chronic disease and counter negative factors in the environment [12]. In order to begin to fill this gap, this Special Issue on "Nutrition Among Vulnerable Populations" features papers quantifying dietary intake, nutritional status, access to food and food security, barriers to healthful foods and food security and environmental influences experienced by vulnerable groups with a high prevalence of food insecurity. The following sections summarize the findings of the four papers on children [13–16], three papers on adults [17–19] and three papers featuring studies of families or households (Figure 1) [20–22].

**Figure 1.** Populations sampled and topical areas of studies included in the Special Issue "Nutrition among Vulnerable Populations".

The diet, health and environmental associations linked with food insecurity or low resources among vulnerable child populations are featured in papers including samples drawn from rarely investigated young children living in Hawai'i, Guam and the Midwestern U.S., while a sample of children and adolescents included in the National Health and Nutrition Examination Survey (NHANES) provided nationally representative contrasts of the diets of food secure and insecure children. Starting with a national scope, the foods and beverages and food groups that were most frequently consumed and contributing most to energy among U.S. children ages 6 to 11 years and 12 to 17 years who were living in situations of food security and food insecurity among household children were determined and compared in a study by Eicher-Miller et al. [14] using NHANES data. Results showed that both the frequency and energy contributions of beverages (including diet, sweetened, juice, coffee and tea) were significantly greater among food insecure compared with food secure children ages 12 to 17 years who had significantly more frequent water intake, while beverage and mixed dish frequency were higher among food insecure children ages 6 to 11 years compared to food secure children who exhibited higher frequency and energy from snacks [14]. Dietary differences by food security status among infants were also investigated by Campbell et al. [13] in a sample from Hawai'i. Surprisingly, findings showed that Native Hawaiian, Pacific Islander and Filipino infants ages 3 to 12 months from food insecure households consumed foods from more food groups and consumed fresh foods on a greater proportion of days compared with infants from food secure households [13]. A community-based sample of children 2 to 8 years old from Guam were the focus of another study evaluating health, lifestyle and dietary intake [15]. Approximately 80% were receiving food assistance, 51% experienced food insecurity and 27.4% were affected by overweight and obesity. Compared with children who had a healthy weight, children who were overweight and obese were more likely to have educated caregivers and to have a higher intake of sugar-sweetened beverages [15]. These dietary and demographic associations with poor health outcomes among young children are important factors to consider in health and food security-promoting interventions. However, broad, environmental-level influences may also be linked with the health and development of young children. The food environment is conceptualized as the availability, affordability and accessibility of grocery stores or other food retail outlets that promote a healthful diet [23]. Parent reports of the community food environment of children ages 3 to 5 years from a Midwestern U.S. state showed that children living in higher quality community food environments had better cognitive ability, specifically executive function, compared with children living in lower quality community food environments [16]. Insights from these child-focused papers contribute new information on the environmental, demographic, lifestyle and behavioral factors of vulnerable groups that influence nutrition, health and development.

Advances in knowledge of the nutrient intake and health risks associated with food security along [17,18] with early effects of the coronavirus pandemic [19] among U.S. adults are featured separately in three articles. Total usual micronutrient intakes from foods, beverages and dietary supplements were compared to the dietary reference intakes among U.S. adults ≥19 years by sex and food security status using nationally representative data from the NHANES [17]. Results showed that both male and female adults living in food insecure households had a higher risk for inadequate intakes of magnesium, potassium and vitamins A, B6, B12, C, D, E and K, while food insecure men also had a higher risk for inadequate phosphorous, selenium and zinc. The risk of inadequacy was not different by food security status for nutrients, calcium, iron (determined in men only), choline or folate. However, the risk for exceeding the tolerable upper intake level was greater among some dietary supplement users [17]. Micronutrient inadequacy may contribute to the risk for chronic disease and poor health, especially when experienced over years into later adulthood [23]. The association of household food insecurity among low-income adults ages 20 to 65 years with cumulative biological risk, a measure of the body's physiological response to chronic stress, was investigated, similarly using NHANES data in a study by Leung et al. [18]. Results showed that women with food insecurity had higher cumulative biological risk scores and higher odds of elevated biological risk, while associations were not observed among men. The authors hypothesized that the chronic stress of food insecurity may facilitate the association with chronic poor health outcomes for women [18]. Another national, although not representative, sample of low-income (<250% of the federal poverty line) U.S. adults ≥18 years old completed a web-based survey to determine the early impact of the COVID-19 pandemic, offering a critical first look at how low-income families are coping with economic and lifestyle changes [19]. Approximately 44% were food insecure, and were significantly more likely to report basic needs challenges compared with food secure adults, with the group experiencing very low food security reporting the most severe difficulties. Food insecure compared with food secure adults were more vulnerable to the economic, dietary and health risks of the pandemic [19]. These current and ongoing effects of the pandemic may compound the micronutrient and cumulative biological risk disparities discovered and documented in these Special Issue articles on U.S. adults.

Clearly, there is a need for interventions that apply knowledge of the barriers, nutrition, health and environmental risks to improve food security and health among low-resource populations. Three studies in this Special Issue focus on interventions or behaviors that may be promoted in future interventions among low-resource families [20–22]. A sample of families with young children in Head Start from a rural area of a northern U.S. state was used to investigate the association of food resource management behaviors, food resource management self-confidence and financial practices with household food insecurity [20]. The participants with high food resource management self-confidence had significantly lower odds of household food insecurity; the inclusion of food resource management self-confidence promotion in nutrition education interventions for the low-resource population may assist management of food dollars to improve household food insecurity [20]. Nutrition education programs like the Supplemental Nutrition Assistance Program Education (SNAP-Ed) have been shown to improve food security and may integrate food resource management self-confidence building to potentially increase the magnitude or sustainability of those changes. Eicher-Miller et al. investigated the characteristics of SNAP-Ed program delivery to determine their role in SNAP-Ed's intervention effect on food insecurity. In addition, the role of participant co-participation in food assistance programs like SNAP was also investigated as a mediator or moderator to food security change due to SNAP-Ed as an intervention [21]. Results of this secondary analysis of data from a longitudinal randomized controlled trial of SNAP-Ed among women ≥18 years from households with children in a Midwestern U.S. state showed that neither variation of program delivery characteristics nor participation or changes in participation in food assistance programs, associated with the impact of SNAP-Ed on change in food security over time, meaning SNAP-Ed directly improved food security among participants [21]. Other interventions among low-income and food insecure participants include incentives to encourage improved fruit and vegetable intake. A scoping review of fruit and

vegetable incentive-based interventions was completed to determine structural factors that influenced program effectiveness [22]. Eighteen of the 19 studies reported a positive impact on either participant fruit and vegetable purchases or intake, and most were located at farmers' markets and offered an incentive in the form of a token, coupon or voucher. The summative knowledge may further inform the design, implementation and success of future fruit and vegetable interventions targeted to improve nutrition among low-income populations [22].

In conclusion, the articles in this Special Issue address dietary intake, behaviors and health among low-resource and food insecure groups. Some of the studies feature populations that have not traditionally been included in research and fill gaps, informing knowledge of the characteristics, lifestyles and environments of these groups. Others feature results representative of vulnerable groups in the U.S. population. These contributions may inform future interventions on food security and dietary intake to incorporate confidence-promoting aspects, an evaluation of the program and participation factors of nutrition education interventions, and a summary of the structural factors of successful fruit and vegetable incentive programs. This Special Issue advances knowledge to improve food security and health among vulnerable U.S. populations.

**Funding:** This work is/was supported by the USDA National Institute of Food and Agriculture, Hatch project 1019736, by the National Institute on Minority Health and Health Disparities of the National Institutes of Health (U54MD00760), and the HMSA Foundation Community Fund grant #CF-021803.

**Conflicts of Interest:** Unrelated to this submission, HAE-M has served as a consultant to Colletta Consulting, Mead Johnson, the National Dairy Council, the Indiana Dairy Association and the American Egg Board. She has previously received travel support to present research findings from the Institute of Food Technologists and the International Food Information Council. However, the funders listed above (USDA) had no role in this editorial. MKF has no conflicts of interest to report.

#### **References**

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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*


**2.5. Lateral profiling of threshold voltages, interface traps, and oxide trapped charge**

220 Dielectric Material

proportional to the strength of the interaction.

**Figure 5.** Principles of IETS technique [22].

degrade the channel mobility.

(b) reverse bias (gate electrode negative) [22].

to the characteristic energy of the inelastic interaction, and the area under the peak is

In a typical MOS sample, there are more than one inelastic mode, as a wide variety of inelastic interactions may take place, including interactions with phonons, various bonding vibrations, bonding defects, and impurities. Figure 6 shows an actual IETS spectrum taken on an Al/HfO2/Si sample, where the features below 80 meV correspond to Si phonons and Hf–O phonons, and the features above 120 meV correspond to Hf–Si–O and Si–O phonons. The significance of this IETS spectrum is that it confirms the strong electron–phonon interactions involving optical phonons in HfO2, and that the Hf–O phonons have very similar energy range as Si phonons which we know are a source of scattering centers that

**Figure 6.** IETS for HfO2 on Si under different bias polarities: (a) forward bias (gate electrode positive),

Lateral profiling is a charge-pumping technique that enables one to profile the lateral distribution of threshold voltages of a MOSFET, and the lateral distributions of interface traps and oxide trapped charge generated by hot-carrier damage [23-24]. Figure 7(a) shows the Icp–Vh curves for the source (curve 1) and the drain junction (curve 2) prior to hot-carrier damage, from which one can obtain the threshold voltage distributions near the two junctions (Figure 7(b)) using the Vh-Vt(x) relationship as described in [25, 26]. Then a channel hot-carrier (CHC) stressing for 300 s to damage the device is used. Comparing curves 2 and 1 in Figure 8(a), one can see that the CHC stressing is not only generated Nit but also caused by positive charge inside the insulator gate, Qot. Therefore, one must neutralize this Qot before proceeding, and this was accomplished by a light hot electron injection as shown by curve 3 in Fig. 8. Note that this step did not cause any increase in Nit as evidenced by the unchanged Icp,max.

**Figure 7.** (a) Single-junction charge pumping curves measured either with the source floating (curve 1) or with the drain floating (curve 2). (b) Local Vt distribution across the channel as deduced from the data in (a).(From Reference [22])

These three Icp curves were then used to extract the Nit(x) from the difference between curves 3 and 1 at a given Vh, and Qot(x) , from the voltage shift between 2 and 3 at a given Icp (Figure 8(b)).

Electrical Characterization of High-K Dielectric Gates for Microelectronic Devices 223

electrons are injected into the substrate. Subsequently, the S/D is reverse biased to create a deep depletion region, which will cause the previously injected electrons in the substrate (those that have not recombined away) to be accelerated across the depletion region and injected into the gate dielectric. This period is called the collecting phase, during which the emitting voltage can control the gate voltage, and large injection current can be achieved with low Vg. Figure 10 illustrates the use of the Vth vs. Ninj curve, obtained by the PASHEI technique, to extract trap parameters. For this particular sample, we obtained a trap density of 2.7 x1012 cm-2, and capture cross-section of 7.7 10-19 cm2, by fitting the trapping theory

**Figure 9.** (a) Schematic description of PASHEI. (b) Pulse sequence for PASHEI. (From Reference [22])

**Figure 10.** Vth vs. Ninj curve obtained by the PASHEI technique, to extract trap parameters. (From

presented by Zafar [29].

Reference [22])

**Figure 8.** (a) Three charge pumping curves measured for the purpose of directly profiling the eraseinduced damage, and graphically illustrating the direct lateral profiling principle. (b) Lateral profiles of both positive oxide charge and interface traps near the source junction, transformed from the three charge pumping curves in (a).(From Reference [22]).