Horticulture

Tissue differences were more revealing at the stage when flavors peak; 4865 transcripts that were significantly higher in skins compared to pulp at 23.2 °Brix were tested for over represented GO functional categories . Some of the top GO categories included photosynthesis, isoprenoid biosynthesis, and pigment biosynthesis . Some of the transcripts with the largest differences between skin and pulp at 23.2 °Brix are β-ketoacyl-CoA synthase , taxane 10-β-hydroxylase , wax synthase, a lipase, an ABC transporter, and phenylalanine ammonia-lyase . The abundance of 5716 transcripts was significantly higher in pulp than skin at 23.2 °Brix . Some of the top GO categories over represented were a variety of transport processes and small GTPase mediated signal transduction . Some of the transcripts with the largest differences in abundance with pulp greater than skin at 23.2 °Brix were polygalacturonase , flavonol synthase, stachyose synthase, an amino acid transporter, a potassium channel , and HRE2 . The transcript abundance of 2053 genes had significantly differential expression across °Brix levels and tissues . The top GO categories over represented in this set involved photosynthesis and phenylpropanoid metabolism, both associated with the berry skin .

Other flavorcentric categories of the 57 categories over represented include aromatic compound biosynthesis, 10 liter drainage pot fatty acid metabolism and alcohol catabolism. This transcript set was further analyzed by dividing into 10 clusters using k-means clustering . The over represented GO categories were determined for each cluster . Eight of the 10 clusters had distinct over represented GO categories; two clusters did not have any over represented GO categories, meaning that the genes in these two clusters were assigned to GO categories of expected proportions when compared to the entire NimbleGen array. Clusters 1, 8, 9 and 10 had a large number of over represented categories. Many GO categories within a cluster are subsets of others in that cluster and were grouped together. For example, cluster 4 had four over represented GO categories, oxygen transport, gas transport, heat acclimation and response to heat. The four categories could be grouped into two, as two are subsets of the others; this is how they were listed in Table 1.It would be impossible to discuss here all the transcript abundance changes detected in these berries. As we were interested in compounds associated with berry flavors as they develop or change in the late stages of berry ripening, we took a more targeted approach for analysis with this in mind.

Berries at 24° Brix are known to be near-optimal for flavor, thus we took a simple approach to look for genes that were peaking around this stage. We found some significant and large increases in transcript abundance between the 22.6 and 23.2 °Brix levels. A group of VviERF6 transcription factor paralogs represented 6of the top 10 transcripts increasing in transcript abundance from 22.6 to 23.2 °Brix in the skin, but not in the pulp . These VviERF6 TFs were also found in Cluster 8 . This is very interesting since many flavor compounds are derived from the skin and ERF TFs are known to be responsive to ethylene, a known fruit-ripening hormone. These VviERF TFs were named ERF105 in the annotation by Grimplet et al. , however they are more orthologous with AtERF6 as determined by a more comprehensive phylogenetic method using many plant species at Gramene . Annotation details of the V1 gene models of the VviAP2/ERF superfamily can be found in Additional file 8 including updated Vvi symbols according to its closest Arabidopsis ortholog as instructed by the Grapevine Gene Nomenclature System developed by the International Grape Genome Program Supernomenclature committee. This renaming of the AP2/ERF super family should facilitate comparative analyses and functions with other species, particularly Arabidopsis. To properly annotate the AP2/ERF super family of Vitis vinifera according to the IGGP Supernomenclature committee instructions, a phylogenetic tree was generated for the AP2/ERF super family of Arabidopsis thaliana and Vitis vinifera using the TAIR 10 and V1 gene models, respectively .

The labeled family classifications were derived from the Arabidopsis naming scheme by Nakano et al.. There are 130 members in the VitisAP2/ERF superfamily in the Pinot Noir reference genome. However, the six paralogs of ERF6 discussed above belong to a Vitis vinifera clade in subfamily IX and are distinctly different or separate from any Arabidopsis subfamily IX ERF TFs . All of these TFs in this clade are orthologs of AtERF6. VviERF6L1 [UniProt: F6I2N8; VIT_16s0013g00900] had one of the most interesting profiles of the 12 members of this clade because its transcript abundance peaked at 23.2 °Brix . Using k-means clustering, VviERF6L1 fell within Cluster 8 with 369 transcripts, including five additional VviERF6 paralogs. The top GO categories associated with Cluster 8 were genes associated with terpenoid metabolism and pigment biosynthesis . Other interesting flavor associated categories included fatty acid and alcohol metabolism . Representative transcripts from Cluster 8 that were correlated with the transcript abundance profile of VviERF6L1 can be seen in Figure 4. These are ACC oxidase, which is involved in ethylene biosynthesis; a lipoxygenase, part of a fatty acid degradation pathway giving rise to flavor alcohols such as hexenol; α-expansin 1, a cell wall loosening enzyme involved in fruit softening, and two terpene synthases, which produce important terpenes that contribute to Cabernet Sauvignon flavor and aroma. The high similarity of these transcript profiles indicates that ethylene biosynthesis and signaling may be involved in the production of grape aroma. Supporting this argument, two recent studies have shown that a tomato ERF TF , falling in the same ERF IX subfamily, has a strong effect on ethylene signaling and fruit ripening. The transcript abundance of AtERF6 in Arabidopsis is strongly increased by ethylene, which is triggered by the MKK9/MPK3/MPK6 pathway. The transcript abundance of VviMKK9 in the Cabernet Sauvignon berries was higher in the skin than the pulp, but there were no significant differences for VviMPK3 or VviMPK6 . This is not too surprising since AtMKK9 activates AtMPK3 and AtMPK6 by phosphorylation. In addition, the transcript abundance of AtERF6 in Arabidopsis increases with ROS, SA, cold, pathogens, and water deficit. There were no visible signs of pathogen infection in these berries. Additional circumstantial evidence for ethylene signaling in the late stages of berry ripening was that the transcript abundance of many VviERF TFs was significantly affected by berry ripening and/or tissue . The transcript abundance of 129 members from the berries was determined to be above background noise levels on the microarray . The expression profiles of the 92 significantly affected AP2/ERF superfamily members were separated into six distinct clusters by hierarchical clustering and indicated that this super family had a complex response during berry ripening . The 12 members of Cluster 1 responded similarly in both the skin and pulp, gradually decreasing with increasing °Brix with a large decrease in transcript abundance at the 36.7 °Brix level. Cluster 2 with 14 members, including 8 members of the VviERF6 clade, had much higher transcript abundance in the skin with a sharp peak at 23.2 °Brix. Cluster 3 had similar profiles in both the skin and pulp with a peak abundance at 25° Brix. Cluster 4 with 7 members was a near mirror image of cluster 2, 25 liter pot with a sharp valley for transcript abundance in the skin between 23 and 25 °Brix. Cluster 5 had 36 members with a steady increase in transcript abundance in the pulp but no substantial increase in the skin until 36.7 °Brix. Finally, in Cluster 6, there were 13 members with a higher transcript abundance in skins compared to pulp. Their transcript abundance increased with increasing °Brix level, but decreased in the skin. The transcript abundance of important components of the ethylene signaling pathway characterized in Arabidopsis and presumed to be functional in grape were also affected by °Brix level and tissue .

Three different ethylene receptors, VviETR1, VviETR2, and VviEIN4 decreased with °Brix level in the skin, however there was very little or no change in the pulp. Likewise, VviCTR1, another negative regulator of ethylene signaling that interacts with the ethylene receptors, decreased between 22.6 and 23.2 °Brix in both the skin and the pulp. The transcript abundance of the positive regulator, VviEIN2, peaked at 25 °Brix in both the skin and the pulp. AtEIN2 is negatively regulated by AtCTR1 and when it is released from repression, turns on AtEIN3 and the ethylene signaling pathway downstream. The transcript abundance of VviEIN3 increased with °Brix level, peaking at 25 °Brix in the skin, and was much higher than in the pulp. Although more subtle, its profile was very similar to VviERF6L1. Derepression of the negative regulators and the increase in positive regulators indicated that ethylene signaling was stimulated during this late stage of berry ripening.The transcript abundance of many of the genes involved in the isoprenoid biosynthesis pathway peaked between 23 and 25 °Brix level, particularly in the skin; this stimulation of transcript abundance continued in both the carotenoid and terpenoid biosynthesis pathways . DXP synthase is a key regulatory step in isoprenoid biosynthesis and its profile was similar to VviERF6L1; its transcript abundance was correlated with the transcript abundance of several terpene synthases in the terpenoid biosynthesis pathway . About 50% of the putative 69 functional terpene synthases in the Pinot Noir reference genome have been functionally characterized. Another 20 genes may be functional but need further functional validation or checking for sequencing and assembly errors. On the NimbleGen Grape Whole-Genome array there are 110 probe sets representing transcripts of functional, partial and psuedo terpene synthases in Pinot Noir . It is uncertain how many may be functional in Cabernet Sauvignon. There were 34 probe sets that significantly changed with °Brix or the °Brix and Tissue interaction effect; 20 of these are considered functional genes in Pinot Noir. Terpene synthases are separated into 4 subfamilies in the Pinot Noir reference genome; they use a variety of substrates and produce a variety of terpenes. Many of these enzymes produce more than one terpene. The top 8 transcripts that peaked in the skin at the 23.2 to 25 °Brix stages were also much higher in the skin relative to pulp . Five of the eight probesets match four functionally-classified genes in Pinot Noir ; these terpene synthases clustered very closely with VviTPS54, a functionally annotated – Linalool/- Nerolidol synthase. VviTPS58, a -geranyl linalool synthase, was also in the cluster. The other two probesets match partial terpene synthase sequences in the Pinot Noir reference genome. The transcript abundance of genes involved with carotenoid metabolism also changed at different °Brix levels and with tissue type . CCDs are carotenoid cleavage dioxgenases and are involved in norisoprenoid biosynthesis. The transcript abundance of VviCCD1 changed significantly with °Brix level and was higher in skin than pulp, except at 36.7 °Brix. Likewise, the transcript abundance of VviCCD4a and VviCCD4b changed significantly with °Brix level, but was higher in the pulp than the skin. The transcript abundance of VviCCD4c significantly increased with °Brix level, but there were no significant differences between tissues. VviCCD1 and VviCCD4 produce β- and α-ionone , geranylacetone , and 6-methyl-5-hepten-2-one in grapes. The transcript abundance of VviCCD8 significantly increased with°Brix level and was higher in pulp than skin. Phytoene synthase, which was also increased in the skin compared to the pulp , and VviCCD1, have been associated with β-ionone and β-damascenone biosynthesis. Other important grape flavors are derived from the fatty acid metabolism pathway and lead to the production of aromatic alcohols and esters. The transcript abundance of many genes associated with fatty acid biosynthesis and catabolism changed with °Brix level . In particular the transcript abundance of a number of genes were correlated with the transcript abundance of VviERF6L1 including VviACCase, Acetyl-CoA carboxylase; KAS III ; VviOAT, ; VviFAD8; ; VviLOX2 and VviHPL . The transcript abundance of alcohol dehydrogenases was affected by tissue and °Brix level . Some ADHs are associated with the production of hexenol and benzyl alcohol. Methoxypyrazines give herbaceous/bell pepper aromas. They are synthesized early in berry development and gradually diminish to very low levels at maturity. Nevertheless, humans can detect very low concentrations of these aroma compounds. Four enzymes, VviOMT1, VviOMT2, VviOMT3 and VviOMT4 , synthesize methoxypyrazines.

The Chern number is just a property of a band and does not come with an energy scale, so there is no reason to expect to encounter Chern bands only at low temperatures. Indeed, bands with finite Chern numbers have been shown to support quantized Hall effects in graphene quantum Hall devices at room temperature and high magnetic fields, as illustrated in Fig. 8.5A,B. The energy scale in a Chern magnet is set by the band gap produced by magnetic interactions. So if we’d like to know what the maximum temperature at which we can expect to find Chern magnets is, we need to think about the energy scales of known magnets. Magnetism is an interaction-driven electronic phase, and interaction-driven phases almost always melt at sufficiently high temperatures. However, among interaction-driven electronic phases ferromagnetism is particularly stable. Many common transition metals, including iron, cobalt, and nickel, support ferromagnetism into the range 600-1200 K, and all of these have found applications in a variety of electronic technologies as a result. These are of course all three dimensional crystals, vertical farm tower and Chern magnets are two dimensional crystals. So the next question we can ask is: do two dimensional magnets exist with Curie temperatures as high as room temperature?

The answer turns out to be yes, as illustrated in Fig. 8.5C,D. This magnetic system appears not to be a Chern magnet, unfortunately, but the point is that there is nothing in particular stopping a Chern magnet with a Curie temperature above 300 K from existing. The first intrinsic two dimensional ferromagnets were discovered in 2017, so I think it’s safe to say that our field hasn’t yet come particularly close to identifying all possible two dimensional magnets. It’s hard to do an accurate accounting of all of the so-far discovered two dimensional magnets, and it is certainly the case that many of these are are not Chern magnets. But of the two dimensional magnets we have found, a surprisingly large fraction are intrinsic Chern magnets. We know of eight intrinsic Chern magnets stable in the absence of an applied magnetic field in the published literature so far. These are presented, along with a few of their basic properties, in Table 8.1. We have discussed several of these materials in this thesis, but we have also skipped a few,including the only currently known intrinsic Chern magnet in an atomic crystal, i.e., not on a moir´e superlattice: MnBi2Te4. These other materials all also represent areas of active research. Of the Chern magnets we know about, 2/8 have C < 0 with B > 0, so that property might be quite common. Indeed, there’s no particular reason to expect the B > 0 ground state to have one sign of the Chern number over the other as far as I know. It’s worth mentioning that if we ever find one, a room temperature Chern magnet with C < 0 for B > 0 would also have extremely large ∆m, and will therefore likely be switchable, since ∆m increases linearly with EGap.

It is also the case that two of these materials have been observed to be switchable with pulses of electric current, although it is not yet clear if the tBLG/hBN and ABMoTe2/WSe2 Chern magnets share a common current-switching mechanism, or if their respective mechanisms would generalize well to large gap Chern magnets. I think it’s clear that we are in the early days of the study of this class of material systems, and without discovering more Chern magnets there is little we can say with much generality. All of this is to say that I don’t think it’s crazy to expect to discover Chern magnets at much higher energy scales than we have already encountered, and that should we ever find such a system, there are a variety of intriguing technological applications for which this class of material systems could be exploited.Over the course of my PhD, four nanoSQUID microscopes were proposed, and construction began in some form on all of them. By the time I left we had finished three of these microscopes. The first nanoSQUID microscope we completed was inserted into a bath of liquid helium and could operate at 4 K. The CrI3 magnetic imaging campaign was performed in this system. The second nanoSQUID microscope had a pumped He-4 evaporative cooling pot, and could reach temperatures of 1.5 K. The tBLG/hBN Chern magnet transport measurements, the tBLG/hBN Chern magnet imaging measurements, and the AB-MoTe2/WeSe2 Chern magnet imaging measurements were all performed in this system. The third nanoSQUID microscope had a closed cycle He-3 sorption pump cooling system, and could reach 300 mK. The fourth and final microscope remains under construction, and is designed to operate inside of a dilution refrigerator.

Pictures of several of these microscopes are shown in Fig. 8.6. Acoustic isolation chambers and the 300 mK system are not shown. All nanoSQUIDs have liquid He-4 baths for primary stage cooling, and all are mounted on several thousand pound vibration isolation tables floating on air legs to protect the nanoSQUID sensors from mechanical and acoustic shocks close to the surface. The nanoSQUID sensor circuit is fairly simple, with only one important non-standard circuitelement in it, other than the nanoSQUID itself of course. This is the series SQUID array amplifier. Current is forced into the nanoSQUID sensor in parallel with a shunt resistor of comparable resistance to the nanoSQUID sensor in the voltage state, which is generally a few Ohms. Current through the nanoSQUID side of the circuit is inductively coupled to a series of identical SQUIDs. These SQUIDs in series generate a large voltage, which is detected at room temperature. Current is forced through a feedback coil to maintain constant flux through the SQUIDs in series. This allows the circuit to maintain sensitivity over a wide range of currents . This current amplification circuit has good current sensitivity and enormous dynamics range, easily able to accommodate the several hundred µA necessary to reach the critical current of the nanoSQUID sensor. There are a lot of things that make scanning probe microscopy tough relative to other techniques for performing microscopy. One particularly challenging issue is navigation of the sensor to the sample. Those experienced with optical imaging might be spoiled by a contrast mechanism that is sensitive to a ton of different phenomena- the nanoSQUID can only see local gradients in magnetic field and temperature, vertical plant tower and those are rare unless you have intentionally built structures and devices that generate them for use in navigation. In particular, large thermal gradients and variations in local magnetic field aren’t general properties of surfaces, so it’s very easy to blunder a nanoSQUID sensor into a surface without ever seeing it coming! Experiments are thus much safer and more expedient if we can provide the nanoSQUID sensor with topographic feedback- i.e., some way of detecting surfaces without crashing into them and destroying the sensor. We did this using shear force microscopy, which is a form of atomic force microscopy, or AFM. There is nothing particularly atomic about this contrast mechanism in the nanoSQUID microscope- we don’t have nearly that much resolution- but it is incredibly useful for navigation because it allows us to safely and reliably detect surfaces without destroying the SQUID. Researchers and companies building scanning tunneling microscopes will often accomplish this by gluing their sensor, which is a microscopic metallic wire, onto a piezoelectric tuning fork and then exciting the tuning fork at its resonant frequency. This is a good strategy, but it must be modified for use with the nanoSQUID sensor, because the nanoSQUID sensor is considerably more massive than scanning tunneling microscope wires, so it cannot be glued onto the tuning fork without destroying its quality factor. We preserve the tuning fork’s quality factor by instead pressing a piezoelectric tuning fork against the side of the nanoSQUID sensor and performing shear force microscopy instead of tapping mode microscopy.

The glass micropipettes serving as substrates for the nanoSQUID sensors are so thin that they bend easily when pressed agains the tuning fork, and this keeps them in mechanical contact with the fork. An optical microscope image of a nanoSQUID sensor pressed against a tuning fork is shown in Fig. 8.8A, and the resonant frequency of the piezoelectrically driven tuning fork is shown in Fig. 8.8B, with a fit to a Butterworth Van-Dyke model. A phase-locked loop and PID feedback system together allow us to approach the surface with the nanoSQUID sensor, detect it without crashing into it and destroying the tip, and maintain feedback while scanning. Schematics of this assembly are shown in Fig. 8.9. A calibration of the scan range and height of the nanoSQUID AFM is shown in Fig. 8.10, with a comparison to a Bruker Icon AFM displayed as well.By far the most common experimental campaign for the nanoSQUID microscope during my time in Andrea’s lab involved being handed a sample fabricated primarily for transport or capacitance measurements, with little consideration afforded to the viability or ease of a scanning probe microscopy campaign on the sample. I think this is fairly common in scanning probe microscopy, and it often means that we need to get sensors to samples without much in the way of navigation infrastructure. For this reason the vast majority of nanoSQUID microscopy campaigns start with thermal navigation. Before cooling down the nanoSQUID microscope, an attempt is made to align the nanoSQUID sensor with the heterostructure under an optical microscope, but the nanoSQUID sensor often still starts several hundred microns away from the sample. Once the system is cold, we generally proceed by injecting a few mBar of helium gas into the sample chamber. This facilitates thermal transport between the nanoSQUID sensor and the sample. We then run an AC current through the sample, heating it and generating an AC temperature distribution. The nanoSQUID sensors are excellent thermometers as well as magnetometers, so we can use this thermal gradient to navigate to the sample. An image of the resulting distribution of temperature over the device is shown in Fig. 8.13A. Some of the details are described in a later section, but in summary this technique works surprisingly well- we can usually find samples even several millimeters away from the nanoSQUID sensor using this technique. Once the nanoSQUID is reasonably close to the sample, it is usually necessary to pump out the heat exchange gas before attempting magnetic imaging, since thermal contrast can produce large backgrounds. After the heat exchange gas is removed, further navigation must proceed by imaging the magnetic fields produced by applied current through the Biot-Savart effect, as illustrated in Fig. 8.13B. There are a few engineering challenges associated with fabricating nanoSQUID sensors. I will briefly describe a particularly challenging one in this section. Many of the best elemental superconductors are soft, heavy metals with low melting points like lead and indium. As any person who has spent some time in an experimental physics laboratory knows, solder doesn’t wet too many materials well, and it certainly doesn’t wet glass, so these metals tend to form droplets when deposited onto glass substrates. To form a uniform film, the superconducting metal must freeze instantly upon landing on the glass micropipette. To make sure this occurs, we must cryogencially cool the glass micropipettes while evaporating the superconducting metal onto them. This process involves specialized machinery that is covered in great depth in other documents and publications, so I won’t discuss it here. However, I do want to discuss the nature of the failure modes of this process. When liquids don’t wet surfaces well, they dewet into droplets, and these droplets tend to get more spherical and less film-like the worse they wet the surface. If this process is allowed to proceed to its conclusion before deposited metal solidifies, the resulting films won’t be connected at all, and your nanoSQUID circuit will be open. If the substrate is cold enough, the resulting film will at least be continuous, and it is likely that you will get a nanoSQUID. However, the formation of droplets is impossible to completely stop, especially near the edges of films and on the oblique surfaces of the nanoSQUID sensor . These droplets generally won’t short the sensor, but the nanoSQUID sensor is so small that electrons can reach these droplets through tunneling processes. Whenever droplets form between the two superconducting contacts on the nanoSQUID sensor electrons can tunnel between the contacts through the droplet, with the droplet functioning as a quantum dot.

This procedure will also produce strong magnetic signals if magnetism couples strongly to current in the two dimensional crystal we’re studying, and this will be the case in several of the systems we will discuss later. As I mentioned either, the nanoSQUID is well-thermalized to its environment, and its properties are quite sensitive to temperature, so we can use the nanoSQUID microscope as a sensitive nanoscale thermometer. I will not present any scientific conclusions based on scans performed using this technique, but it is a powerful capability and it is also useful more practically for nanoSQUID navigation, which is discuseed further in the appendix. Electron spin is a fundamentally quantum mechanical property; it can be more or less understood using analogies to classical physics, but it also has some properties that don’t have simple classical analogues. Spin can be understood as a quantized unit of angular momentum that an electron can never be rid of. Although an electron is, as far as we know, a point particle, this unit of angular momentum couples to charge and produces a quantized electron magnetic moment, which we call the Bohr magneton, µB. Electron spins both couple to and emit local magnetic fields, stackable flower pots and they are orthogonal to the electronic wave function- changing an electron’s wave function will not under normal circumstances influence its spin, and vice versa.

Electrons are fermions; they obey the Pauli exclusion principle, which states that no two electrons can be placed into the same quantum state. The simplest consequence the existence of electron spin has is the fact that electronic wave functions can fit two electrons instead of one, because an electron can have either an ‘up’ spin or a ‘down’ spin. We say that electron spin produces an energetic degeneracy, because each electronic wave function can thus support two electrons. Electron spin is not the only degree of freedom that can produce energetic degeneracies; we will discuss a different one later. All of the above arguments apply for electron spin in condensed matter systems as well, and we can expect every electronic band to support both spin ‘up’ and spin ‘down’ electrons. These arguments say nothing about interactions between electrons, and all of the physical laws we normally expect to encounter still apply. In particular, electrons of opposite spin can occupy the same wave function, but a pair of electrons have like charges, so they repel each other. There is thus an energetic cost to putting two electrons with opposite spin into the same wave function, and this cost can be quite large. This consideration is outside the realm of the physical models we have so far discussed, because electronic bands in the simplest possible picture are independent of the extent to which they are filled. We are introducing an effect that will violate this assumption; the energies of electronic bands may now change in response to the extent to which they are filled.

In particular, when an electronic wave function is completely filled with one spin species , it will remain possible to add additional electrons with opposite spins, but there will be an additional energetic cost to doing so. It is important to be precise about the fact that the displacement of the unfavorable spin species upward in energy occurs after the wave function is filled with its first spin. As a result, which spin species gets displaced upward in energy is arbitrary, and is determined by the spin polarization of the first electron we loaded into our wave function. This is an example of a ‘spontaneously broken symmetry,’ because before the addition of that first electron, the two spin species were energetically degenerate, and after the band is completely filled with both electron species, they will again be energetically degenerate. All of the above arguments apply to localized electronic wave functions and do not say anything specific about condensed matter systems, which involve many separate atoms that each support their own wave functions. A similar but somewhat subtler argument applies to electronic wave functions on adjacent atoms in condensed matter systems. When electronic wave functions on two adjacent atoms overlap, the structure of the delocalized electronic band that will emerge from them when they hybridize depends strongly on their relative spin polarization. When electrons on adjacent atoms have the same spin, the Pauli exclusion principle will prevent them from overlapping, thus minimizing their Coulomb interaction energy.

When electrons on adjacent atoms have opposite spins, the Pauli exclusion principle doesn’t apply, because the two electrons are already in different quantum states, and they can overlap. This produces a larger interaction energy for arrangements wherein electrons on adjacent atoms have antialigned spins . Like all qualitative rules there are exceptions wherein other energetic contributions are more important, but this argument applies to a wide variety of condensed matter systems. These systems are known as ‘ferromagnets.’ They have interaction-driven displacements of minority spin bands, are at least partially spin polarized, and have electron spins that are largely aligned with each other. Both of these energy scales, the ‘same-site interaction’ and the ‘exchange interaction’ respectively, can be quite large in real condensed matter systems. The presence of these effects can produce a variety of phenomena. The displacement of a spin subband upward in energy can produce partially spin-polarized metals , fully spin-polarized metals which we call ‘half-metals’ , and spin-polarized insulators which we call ‘magnetic insulators’ . Examples of each of these kinds of systems are known in nature, and all of these phenomena represent manifestations of magnetism. In principle one must perform calculations to determine whether magnetism will occur in any specific system. In practice there exist good rules of thumb for making qualitative predictions. Same-site interactions and exchange interactions minimize energy by minimizing the number of minority spin species present in a crystal, and putting the electrons that would otherwise have occupied minority spin states into majority spin states. Of course, this process always requires that the system pay an additional energetic cost in kinetic energy, because those previously unoccupied majority spin states started out above the Fermi level. The competition between these energy scales determines whether magnetism will occur in any particular material. It follows that systems with a multitude of quantum states with very similar energies in their band structure will be more likely to form magnets; to put it more precisely, we are looking for situations in which, near the Ferm level at least, E = C, where C is some constant. We can say that under these circumstances, the energies of electrons in the crystal are independent of their momenta. We can also say that we have encountered a large local maximum or even a singularity in the density of states. We sometimes call this the ‘flat-bottomed band condition,’ or just the ‘flat band condition’ , and it can be made quantitative in the form of the Stoner criterion. Magnetism is perhaps the simplest phenomenon that can be understood in this context, but it turns out that this argument applies very generally, tower garden and physicists expect to find a variety of interesting phenomena dependent on electron interactions whenever we encounter these situations. It is important to be specific about what we mean by a flat band here: we expect to encounter magnetism whenever an electronic band is locally flat- it is fine for the band to have very high bandwidth as long as it has a region with E ≈ C. These systems will tend to produce magnetic metals. When we encounter bands that are truly flat- i.e., they have both weak dispersion and small bandwidths- we are more likely to encounter magnetic insulators, as illustrated in Fig. 2.3. Most electrons in condensed matter systems are not moving at relativistic velocities. However, in the outermost valence shells of very large atoms , electrons can end up in such high angular momentum states that their velocities become relativistic.

We can thus expect electrons in bands formed from orbitals supported by heavy atoms to respond to local electric potential variations as if they provide a local magnetic field. This phenomenon is known as spin-orbit coupling, and it provides a mechanism through which the energy of an electron spin can couple to the electrostatic environment inside of an atomic lattice. Predicting the global minima in energy as a function of spin orientation is very challenging, but it is often true that a discrete set of minima exist, and of course they must obey the symmetries of the atomic lattice. For this reason in many magnetic materials there is a discrete set of magnetic ground states defined by axes along which the electron spin can point. It is very often the case that there exist two global minima in energy that are anti-parrallel along an axis of high symmetry; when this is the case, we say that the system is an Ising ferromagnet. The axis along which the ground state spin orientation points is called the ‘easy axis.’We are now ready to discuss a real magnetic system. Chromium iodide is a two dimensional magnetic insulator. Systems like chromium iodide have properties that are easy to understand in the context of the models we have so far discussed: strong on site interactions and exchange interactions produce full spin polarization, an interaction-driven band gap, and aligned magnetic moments within a single layer. As a result, these systems are electrical insulators. They support magnetic domain dynamics, and there is a temperature TC above which they cease to be magnetized , although they remain insulators far above that temperature. Extremely weak out-of-plane bonds produce highly anisotropic cleavage planes and make it relatively easy to prepare atomically thin crystals mechanically. As in other systems, this does not mean we will always be studying monolayers of the material. Bilayers, trilayers, four-layer crystals, and even thicker flakes can all have properties that differ significantly from those of a monolayer, often for reasons that we can understand, and CrI3 is no exception. Although it isn’t particularly relevant to the physics of magnetism, it’s worth mentioning that all of the chromium halides are highly unstable compounds, and decompose in a matter of seconds when exposed to air or moisture. These materials are difficult to study under normal circumstances, but two dimensional crystalline samples can be prepared inside of an inert-atmosphere glovebox. They can also be sandwiched, or ‘encapsulated,’ between other two dimensional crystals. Two dimensional crystals are so flat that this process produces an air- and water-proof barrier and protects the encapsulated crystal from degradation in atmosphere, facilitating easy measurements with tools like the nanoSQUID. The crystalline structure of CrI3, projected onto a two-dimensional crystal, is visible in Fig. 2.6A. Unlike graphene, CrI3 has two different kinds of atoms in its unit cell; the chromium atoms are responsible for the magnetic moments producing magnetism. CrI3 has fairly strong spin-orbitcoupling, and thus strong Ising anisotropy, with magnetic moments pointing out-of-plane . Most of the other chromium halides also support magnetic order, although the precise nature of each of their ground states differs somewhat. Both CrI3 and CrBr3 have ferromagnetic in-plane interactions and strong Ising anisotropy, but CrI3 has antiferromagnetic out-of-plane interactions, meaning that in the magnetic ground state of the crystal adjacent layers have their spins antialigned . Interestingly, CrCl3 also seems to have ferromagnetic in-plane interactions, but it is likely that it is not an Ising or easy-axis magnet, and instead has its spins pointed in the in-plane direction and thus free to rotate. It is evidently the case that although these systems are structurally very similar and all have strong spin-orbit coupling, their magnetic interactions and magnetocrystalline anisotropies vary wildly in response to modest differences in their electronic structure. As a result of all of the arguments discussed previously in this chapter, a CrI3 monolayer has finite magnetization even in the absence of an applied magnetic field, and its magnetic order experiences hysteresis in response to variations in the applied magnetic field, as illustrated in Fig. 2.6D . Antiferromagnetic interactions between adjacent layers in CrI3 mix in an interesting factor that can be easily understood: flakes with an even number of layers have no net magnetization in the absence of an applied magnetic field, but develop finite magnetization at higher magnetic fields as the applied magnetic field overwhelms interlayer interactions and realigns each layer in turn with the ambient magnetic field .

As well as direct interaction and disturbance, avoidance of interspecific chemical cues and resource competition have the potential to alter pollinator foraging movements. Global human population growth is putting greater pressure on agricultural production. There is concern over how to meet the increasing demand for food, while at the same time safeguarding ecosystems and biodiversity. In the future, land currently under agricultural production will have to be more intensively managed to increase yields and/or more land will have to be converted to agriculture. Given the negative impact agriculture has already had on biodiversity, it is important that future steps to increase production be made environmentally sustainable. In the last 50 years, the fraction of agricultural production requiring biotic pollination has more than tripled. When compared with crops that are not pollinator-dependent, those that are moderately pollinator-dependent have shown slower growth in yield and faster expansion in area from 1961 to 2006. Almond is a mass-flowering, varietally self-incompatible crop species, hydroponic nft gully highly dependent on biotic pollination. Almond orchards are generally planted with alternating rows of two or more varieties.

Planting a single variety per row facilitates harvest, but complicates pollination because pollen must be transferred between rows to achieve fruit set. To allow for management activities, trees between rows are further apart than those within the same row . Apis mellifera tend to forage within a tree and then move down the same row, probably because less effort is required to move to the next tree in the same row or because the rows act as visual markers that influence movement. This foraging pattern means A. mellifera tend to move more incompatible pollen, limiting their pollination effectiveness. In almond, we investigated whether the presence of nonApis bees affected the behaviour and pollination service of the dominant pollinator species, A. mellifera. Often almond orchards are isolated from natural habitat and non-Apis bees can be completely absent. Therefore, we were able to compare A. mellifera behaviour and pollination effectiveness in diverse bee communities with orchards lacking non-Apis bees. Here, we refer to pollinator effectiveness as the probability an ovule is fertilized following a single visit. We complemented our intensive field sampling with observations in a controlled cage environment, where A. mellifera were introduced along with the blue orchard bee Osmia lignaria Say. We hypothesized that where non-Apis bees were present, such as in sunflower, interspecific interactions would cause A. mellifera to more frequently move between rows. We further hypothesized that an increase in between-row movements by A. mellifera would increase their pollination effectiveness and increase fruit set .

Our findings show that increased pollinator diversity can synergistically increase pollination service through species interactions that alter the behaviour and resulting functional quality of a dominant pollinator species. This highlights a largely unexplored facilitative component of the benefit of biodiversity to ecosystem services. Total bee visitation rates were similar between orchard types, and the visitation rate of A. mellifera was lower in orchards with non-Apis bees. However, A. mellifera pollination effectiveness was greater and fruit set was higher when non-Apis bees were present. Apis mellifera increased their proportion of movement between tree rows when non-Apis bees were present, thereby improving pollination effectiveness. More successful pollen tube growth translated into significantly higher fruit set in the orchards where non-Apis bees were present. The greater proportion of between row movements by A. mellifera individuals most probably resulted in the deposition of more compatible pollen, since pollen from the same variety generally does not set fruit . This synergistic effect of the presence of non-Apis bees suggests that maintaining biodiversity in agricultural ecosystems could provide unrecognized benefits, and it offers exciting opportunities for the integration of more diverse pollination systems to improve the longer-term sustainability of crop production for almond and similar crops. Unlike in sunflower, very few direct interactions were observed between A. mellifera and non-Apis bees. One potential mechanism for the increased proportion of inter-row movement of A. mellifera in the presence of other non-Apis bees is linked to resource depletion. Because some non-Apis bees can fly at lower temperatures than A. mellifera, and therefore earlier in the day, it may be that if A. mellifera begin foraging and find flowers have already been depleted then they increase the distance of their foraging flights. Alternatively, it may be related to the scent marks left by non-Apis bees on the flowers.

Apis mellifera have been shown to avoid visiting flowers that have been marked by bumble bees, and it may be that as well as avoiding the flowers they also shift their foraging location. Given that A. mellifera tend to move down the same row, if a response to perceived resource competition is to shift foraging locations, this could be expected to involve movement across rows. However, at present the mechanism for the increased between-row movements when non-Apis bees are present is unknown. In the controlled cage environment, the steeper increase in the number of pollen tubes initiated with increasing pollen deposition in the mixed cage and the higher fruit set supports the findings from the open orchards that more compatible pollen is being moved when pollinator communities are diverse. Because there was only one cage per treatment, the data from the cages are only descriptive, but they do support the findings from the open orchards. Future work should attempt to replicate similar treatments. The greater fruit set in the mixed cage when the visitation rate was lower than in the A. mellifera cage may be due to the slightly higher proportion of between-row movements in the mixed cage. Although the differences in movement were not great, the pollen tube data suggest that more pollen was moving between varieties in the mixed cage. The size of the cages limited the opportunity for between-tree flights. This and the relatively low visitation rate of O. lignaria in the cages compared with the open orchards may explain why the difference in pollen tubeformation and fruit set between the cages was less than in the open orchards. The price of California almond in 2011 was approximately $1.79 per pound and production was estimated at 2670 lbs per acre . If a 5 per cent increase in fruit set as calculated from the controlled cage environment translated into an equivalent increase in production, farmers would make $239 more per acre . This estimate from cages may be a lower bound compared with open orchards. Sampling effects and complementarity have been the primary explanations for a positive relationship between biodiversity and ecosystem function. Here, we show a different mechanism, possibly due to interspecific competition, where community composition alters the behaviour of a service-providing organism with a positive knock-on effect for the ecosystem service. Our results show alterations in A. mellifera foraging behaviour when a diverse community of other bees are present, and suggest almond yield can be increased by encouraging wild bees in the orchards. As such, dutch buckets for sale natural habitat near almond orchards should be conserved to protect wild bee communities. The availability of A. mellifera is not predicted to increase at the same rate as demand for their services in agriculture. Thus, increasing the pollination effectiveness of A. mellifera and conserving wild pollinator communities could help increase crop yields. The synergistic combination of A. mellifera and non-Apis bees represents a sustainable way to improve crop pollination services, but the generality of such effects still need to be tested across multiple crop systems.The two coffee species of commercial value, Coffea arabica and Coffea canephora , both originate from Africa; the former has generally preferred taste qualities, and the latter exhibits higher yield and pest resistance . Coffea arabica dominates global coffee landscapes, accounting for 60% of the total coffee volume produced . Although coffee’s center of origin lies in Ethiopia, major global dispersal of the bean occurred when Arab and European traders introduced the beverage to Western Europe in the early 1500s . By the latter half of the 1800s, coffee plantations of both C. arabica and C. canephora flourished throughout the American tropics; by the 1970s, coffee’s cultivation dominated more than 8.8 million hectares of tropical landscapes. Between 1970 and 1990, the global coffee area and average yields increased by 25% , and global production increased by 58% .

Interestingly, although the global area decreased to 10.2 million ha between 1990 and 2010 , production still climbed 36%, which is evidence of an overall intensification in several key countries , coffee abandonment in others , and the rapid expansion of high-yield coffee in new countries . Brazil, for instance, saw a 112% jump in production with only a 12% increase in coffee area between 1996 and 2010, growth spurred by intensification that resulted in an 89% yield increase over that period , and recognition from coffee experts that production there has been highly industrialized . Since the mid-1980s, exports of robusta coffee have increased by 92%, led by a number of Asian countries, with Vietnam being the prime example, exhibiting hand-in-hand increases in both area and intensification . Robusta yields there soared from a historical average of 450 kg per ha prior to the 1950s to 1558 kg per ha by 2004 , more than double the global yield average at the time, which reveals that a species shift alone does not explain yield increases. Given that coffee area decreased globally by 9% between 1990 and 2010, whereas world production increased by 36%, we posit that intensification is one of the major drivers of shifting coffee cultivation practices. A closer look reveals that the shift in production between 1990 and 2010 was regional: 45% of the nations exhibiting decreases were in Africa, whereas Asian countries accounted for 35% of those with increased production . When the first comprehensive studies of coffee and biodiversity emerged in 1996, the top three producing countries were Brazil, Colombia, and Indonesia. Currently, Brazil, Vietnam, and Indonesia top the list, accounting for 57% of the 8.2 million metric tons produced in 2010. In Vietnam alone, the cultivated area increased by 731%, yields by 45%, and the total production by 1102% between 1990 and 2010 . In contrast, the past 20 years reveal coffee area declines exceeding 20% in Ecuador, Colombia, Côte d’Ivoire, Mozambique, Madagascar, Tanzania, and Rwanda . The contrasting and heterogeneous changes in global coffee cultivation result from multiple factors, including region-specific economic development patterns, political conflict, cultural practices, land values, wages, and labor. For example, deforestation accompanied increases in coffee area in Vietnam, Indonesia, Nepal, and Panama . In contrast, in places where the coffee area has declined, such as Costa Rica and Ecuador, the expansion of high-yield agriculture has caused a decrease in coffee prices, which has, in turn, resulted in the abandonment of marginal agricultural lands in combination with increased land prices due to urbanization. Higher land values due to exurbanization often displace coffee cultivation in places like Panama’s Boquete and Chiriquí regions, Costa Rica, and Guatemala— areas now popular as retirement destinations . In a number of countries, waves of political and social instability have reduced investment in coffee cultivation , but sustained global prices after 2005 have spurred expansion in other countries . In other regions, the draw of better urban wages or displacement by other cash crops, such as cacao , has reduced the area of coffee production. Despite variation in global coffee production, the majority of coffee is still produced by smallholders managing less than 10 ha of coffee , as has been documented in Asia and in Africa . Likewise, in Central America, smallholders represent 85% of coffee producers but control only 18% of coffee production lands . In some coffee-producing countries, such as Rwanda, coffee farm sizes are so small that the majority of farms are measured by the number of coffee bushes instead of in ha , whereas in many Mesoamerican smallholder farms, stand densities are as high as 6700 coffee bushes per ha . These patterns in farm size tend to shift, depending on coffee prices and government incentives, as is evidenced in Latin America, where a decrease in the number of large estates and an increase in the number of smallholders and microproducers occurred directly after the 1999 coffee crisis, when coffee prices dropped to century lows . In the Costa Rican coffee district of Agua Buena, the proportion of farmland dedicated to coffee production diminished from 52% to 24% between the years 2000 and 2009, whereas the proportion of pasture land increased from 31% to 50%, largely because of basement level international coffee prices .

The Department of Education projects that enrollments will grow by 0.7 percent in the current fiscal year. The education budget request remarks that “additional funding proposed by the Governor will allow local districts and charter schools to decide how to best improve the education opportunities of their students.” As required by Amendment 23, the inflationary increase to the State Education Fund amounts to $8.9 million. The Department of Education proposed to allocate much of this increase to “categorical programs,” which are educational initiatives tailored to specific groups of students including those with disabilities, ESL students, at-risk students, and gifted and talented students, as well as specialized programs such as technical training, health education, and school transportation. The final budget included funds to support many of these initiatives with a $78.12 million increase in spending from the prior year. This number is below the increase requested in the governor’s budget, hydroponic dutch buckets but does represent an investment in education when many competing priorities exist. In dollar amounts, the proposed increase in higher education funding is similar to that for K-12 education.

Increasing higher education spending by $86.9 million is almost a 10 percent increase from the current fiscal year’s funding level. Among the notable initiatives in the higher education budget are $73.1 million to improve college affordability and outcomes. This includes a $59.1 million allocation to state college governing boards “to address affordability for all students by moderating higher education tuition increases” . Additional funds indirectly distributed to students include $13.9 million for need-based financial aid and work-study programs and $4.0 million for the Colorado Opportunity Scholarship Initiative. The budget also includes lines for new programs such as the $1.5 million proposed for an Emergency Completion and Retention Grant, which is a one-time benefit for students experiencing an unexpected financial hardship, and $5.0 million for a new Occupational Credential Capacity Grant program, which supports “high-demand post-secondary certificate credentials.” The General Assembly elected to fund these college affordability efforts, among other programs, by providing more funding than the governor’s budget requested. The final appropriation for Higher Education was in excess of $1.0 billion, a 12.1 percent increase, which is greater than the governor’s requested increase of 9.7 percent.

In dollar amounts, this increased outlay is among the largest in the approved budget after the $115 million increase approved for Human Services. After K-12 education, health care spending represents the second largest budgetary outlay. Similar to the education budget, legislators approved health care funding at a level slightly less than Governor Hickenlooper’s request. The Department of Health Care Policy and Financing is slated to receive increased General Fund appropriations totaling nearly $100 million, despite an expected decline in the state’s Medicare caseload. Among the new spending initiatives regarding health care include $89.4 million for Medicaid, the Child Health Plan Plus , and the Medicare Modernization Act. Provider rate adjustments proposed in the budget total $10.3 million, which constitute a standard increase of 0.77 percent. The final budget increased health care spending by $81.8 million . In percentage terms, the department with the largest proposed budget increase is Personnel at nearly 17 percent. Funds were also included to hire six full-time employees to assist with the state archives and the address confidentiality program, which assists individuals who have experienced domestic violence. The legislature approved an increase about 12.5 percent, which is the fifth largest increase in the final budget. Departments with an increase in spending in excess of this amount, which were greater than the governor’s proposals, include Treasury, Public Safety, Local Affairs, and Human Services. As seen in Table 3, the largest deviation from the governor’s proposal is the substantial increase in Treasury spending. This increase is primarily attributable to $225 million in approved appropriations from a separate bill as an unfunded liabilities distribution to PERA. Conversely, the department with the largest proposed cut to its budget is Department of Labor and Employment, which the governor’s budget request proposes to reduce by 13.5 percent. The approved budget for Labor and Employment imposes a cut of 8.9 percent, which is the only department’s budget reduced by the General Assembly for this fiscal year.

While not reducing the budget from the prior year, data reported in Table 3 show that legislators ultimately provided less funding than the governor proposed for six departments, while the remaining 13 departments received funding in excess of Governor Hickenlooper’s request. Similar to previous years, other departments with large budgets include Human Services , Corrections , and the courts . The Human Services budget proposed salary increases for staff as well as imposing a 1 percent increase in community provider rates . The budget also proposed nearly $10.0 million in General Fund spending for the Office of Behavioral Health, which provides numerous services including court-ordered health evaluations and outpatient treatment. The proposed correctional budget sought additional funds to address capacity issues since the prison population is projected to increase slightly next year in a modest reversal of an overall downward trend. More than $18.0 million is slated to go toward a variety of health-related programs providing treatment and benefits to current prisoners. Nearly $2 million would be used to hire 14.5 additional full-time employees with the Colorado State Patrol. The department justified the need for additional highway patrol officers in order to reach their goal of equally dividing officer time between reactive and proactive policing. An additional $1.2 million from marijuana tax revenue will fund a special unit within the Colorado Bureau of Investigation to investigate illegal marijuana trafficking.An updated letter from Governor Hickenlooper to the JBC on March 19, 2018 stated that the revised economic forecasting data indicated an additional $548 million available to for state legislators to spend. The governor’s office proposed allocating nearly half of this amount to transportation needs across the state. Transportation funding has been a source of controversy in recent years, as the legislature appeared to have the votes to refer a modest tax increase to voters for consideration in the prior election year. However, a small group of Republicans in the Senate blocked the measure from progressing out of committee. Transportation funding remains one of the key issues on the legislature’s agenda as the session moves toward a close. As in 2017, Governor Hickenlooper’s final State of the State address called for a transportation tax to be placed on the fall ballot. Last year, bato bucket the General Assembly came close to referring a measure to the voters that would increase the state’s sales tax from 2.9 percent to 3.52 percent. This revenue would be used to secure bonds for local and statewide transportation projects. The referendum passed the House with all Democrats voting in favor in addition to four Republicans. Although it appeared likely that the measure would similarly pass in the Senate, it was defeated in committee. Governor Hickenlooper encouraged lawmakers to revisit the transportation tax increase in the current session by arguing, “We need to be even more ambitious. It’s time we look at a long-term solution with a sustainable funding source. There’s broad agreement — across party lines. Coloradans deserve the opportunity to vote on whether we need new resources and where they should come from. It’s time to go to the voters” . The legislature is currently considering a number of alternatives, but it appears likely that the parties will reach some type of funding agreement soon. The current compromise will likely involve a one-time spending allocation to transportation as the governor proposed along with an agreement to defer any ballot measure to increase taxes for transportation funding to 2019 or beyond . However, it is possible that a citizen’s initiative to raise transportation taxes may nonetheless qualify for the ballot. Coloradans are generally not supportive of such proposals. Voters rejected increased cigarette taxes in 2016, and Coloradans twice opposed income and sales tax increases to fund education in 2011 and 2013. Neither outcome was close. In fact, since TABOR was ratified into the state’s constitution in 1992, no proposed tax increase on the statewide ballot has passed with the exception of an increase in cigarette taxes when the electorate approved Amendment 35 in 2004. Voters did, however, approve the tax levels for the new retail marijuana market in 2013.

If the transportation tax initiative is delayed for at least another year, there will be a different governor in office when voters decide whether the addressing state’s transportation problems merit a tax increase. Based upon the party’s positions in the legislature, one would expect that the Republican candidate for governor would oppose a transportation tax increase, while a Democratic candidate would likely campaign in support of the measure. The 2018–2019 state budget provides substantial financial investments in existing and new programs across a wide array of policy areas. Governor Hickenlooper identified education, transportation, and corrections as among the issues worthy of greater funding. A General Fund budget of $11.42 billion exceeds the budget from the prior fiscal year by nearly 8 percent. This increase in spending is made possible by a confluence of factors including a strong economy, federal tax reform, and state budgetary reform from the prior year affecting how the hospital provider fee pertains to the requirements imposed by TABOR. Yet TABOR remains, as its creators intended, a powerful vehicle to limit government revenue and spending. While the state budget increases funding for nearly every state department, a prosperous economy would normally allow for even greater investments in areas of need. Sustained population growth has expanded the tax base, but the state government remains restricted in the amount of revenue it can collect and spend. Citizen initiatives to increase taxes for education and transportation will appear on the ballot in the 2018 midterm election, but Coloradans have rarely demonstrated a willingness to increase taxes. Accordingly, the constraints imposed by TABOR have curtailed even greater investments in state programs that a strong economy and budgetary reform have helped facilitate.A quick history of ecology might begin with the idea that Tansley’s post-WWI botanical surveys ushered in the modern science of ecology, although its basic vision was articulated half a century earlier by Haeckel, in what was a simple statement recognizable even today in its modern manifestation . The application of mathematics to the field was pioneered by the threesome of Lotka, Volterra, and Gausse, in the 1920s and the conceptualization of an ecosystem as a hierarchical energy transfer system by Lindeman in 1942, arguably the foundation on which the Odum brothers successfully launched what we call ecosystems ecology today . The famous Hairston and colleagues paper, perhaps marking the birth of modern community ecology, generated a popular framework that continues generating interesting research questions, all formulated around the central idea that terrestrial communities are regulated by the predators eating the herbivores. Finally, Richard Levins’s lifelong commitment to viewing both ecological communities and ecosystems as complex systems presaged the current popularity of combining recent developments in the field of complex systems with the persistent search for basic framings in community ecology . This is the historical skeleton on which the current essay is built, framed within the Hairston and colleagues hypothesis in the context of Levins’s insistence on a complex systems approach. The most enduring question of Hairston and colleagues remains popular today, indeed takes on new meaning in the context of modern complex systems. Is a community controlled from above or below, from predators or from competition among herbivores for plant resources ? Certainly ecologists have not been credulous to the point of oversimplifying either predator–prey relationships or interspecific competition; however, the simple question “Is control from above or below?” seems a persistent underlying framework for many research programs. The agroecosystem, by its very nature, refuses to entertain this question, at least in its classical form. If the objects of attention are overused by the herbivores , it is not a system that is regarded as one we should allow to persist . Rather, if the system is to persist at all, the herbivores must be regulated from above. Indeed the standard practical agronomic question “How can we control the pests?” if translated into its true ecological meaning would be “How can we cause the system to switch from control from below to control from above?” Control from below has come to mean much more than seems to have been the intent of Hairston and colleagues , especially in light of what we now understand about plant chemistry and its relationship to herbivory .

In a subsequent 2 mo study of the effects of the nutrient bar on CMR markers in individuals across a range of BMIs, only those with low inflammation at baseline as assessed by high sensitivity C-reactive protein < 14.3 nmol/L responded comparably to those in the earlier trial, with not only increased HDL cholesterol and large HDL particles but also a trend toward increased high molecular weight adiponectin and a decrease in other CMR factors at 2 weeks, sustained at 2 months . In particular, a shift in low density lipoprotein particle subfractions toward a less atherogenic profile was evident in the non-inflamed group . Although the participants with overweight or obesity and CRP > 14.3 nmol/L did not show this response, they did experience an upward trend in adiponectin by 2 months. These results suggest that there may be a continuum of metabolic responsiveness to this nutrient supplement that is slowed in the face of the chronic low-level inflammation commonly observed with obesity and insulin resistance. A 6 month study of this nutrient bar in obese adolescents with non-eosinophilic asthma showed improved lung function at 2 months, but favorable movement in cardiometabolic biomarkers only began to emerge at 6 months.It is not known whether more sensitive biomarkers of early metabolic change may be capable of detecting short-term effects of the nutrient bar supplementation in persons at CMR.

Past metabolomics studies in obese adolescents and adults have identified strong positive associations between baseline levels of branched chain, aromatic, sulfur, and gluconeogenic amino acid metabolites and parameters of inflammation and insulin resistance. Similarly, planting gutter elevated levels of specific ceramide species have been shown to associate with inflammation, dyslipidemia and insulin resistance in both adult and adolescent obesity. The relative sensitivity of these biomarkers to reflect moderate changes in dietary intake of polyphenols, essential lipids, fiber and vitamin/minerals, remains incompletely understood. In the present randomized, controlled, non-blinded trial, a two month intervention with exercise and nutrition counseling alone or with nutrient bar supplementation was performed in a high CMR cohort of adolescent /parent adult caretaker family units to determine 1) cross-sectional relationships in both adolescents and adults between traditional CMR biomarkers and amino acid and ceramide metabolites and 2) longitudinal changes within groups in the same CMR biomarkers following the lifestyle +/- nutrient bar intervention.Anthropometric and clinical evaluation: All anthropometric measures were performed in duplicate in the clinical research center and if not within 10% agreement, were repeated a third time. The reported measure is an average of the two closest numbers. Height was measured with a stationary stadiometer.

Weight was measured using a digital electronic scale and the BMI and waist to height ratio were calculated. Waist circumference was measured at end expiration to the nearest mm with a Gulick II Plus tape midway between the lowest border of the rib cage and the upper border of the iliac crest.Blood pressure and Resting Heart Rate: Each was measured in triplicate after 5 minutes sitting quietly with readings taken at least one minute apart. An automatic digital blood pressure monitor was used with cuff size adjusted for arm size. Traditional CMR biomarkers: Fasting blood samples were drawn and processed in the UCSF Benioff Children’s Hospital Oakland Clinical Research Center and sent to ARUP Diagnostic Laboratories for: standard lipid profile [total triglyceride, total cholesterol, and cholesterol within HDL and LDL , glucose, insulin, 25 hydroxy Vitamin D level and CRP. TG to HDL ratio and non-HDL were calculated. Fasting insulin and glucose were used to calculate the Homeostasis Model Assessment of Insulin Resistance Index according to the formula: fasting insulin x fasting glucose /22.5. Lipoprotein particle sub-classes were analyzed by an ion mobility procedure that sensitively and directly measures concentrations of lipoprotein particle subfractions. High molecular weight adiponectin was measured by solid-phase sandwich ELISA . Metabolomic analyses: 1) Targeted analyses of 42 amine-containing metabolites consisting of 20 major amino acids, and secondary metabolites of arginine and cysteine whose levels are sensitive to inflammation and oxidative stress were performed on stored samples preserved at -70o Fahrenheit. Briefly, plasma was acidified with 5% perchloric acid containing 8 stable isotope internal standards. Acid-soluble supernatant was used for strong-cation exchange solidphase extraction to capture cationic amine- containing metabolites. Extracted metabolites were further derivatized with isopropylchloroformate.

Derivatives of metabolites were resolved using Agilent 1260 ultra-high pressure liquid chromatography and eluted with a gradient of water and isopropanol . An Agilent 6490 triple quadrupole mass spectrometer was used to detect resolved analytes and quantify them using authenticated external and internal standards. 2) Sphingolipidomics by electrospray tandem mass-spectrometry by validated techniques was used to identify sphingolipid metabolites, including ceramides.De-identified baseline and study completion data points, paired by participant study ID, were entered into SPSS . Descriptive analyses of the study cohort were summarized and results for Teens and PACs, as well as for combined CONT and INT groups, were compared by unpaired Student t-test. Continuous physical and metabolic variables were tested for normality by examining the skewness, kurtosis and the Shapiro Wilk tests and transformed as necessary before analysis. Most of the variables in our data were normally distributed. Log transformations were conducted for the continuous physical and metabolic variables that were skewed to make them as normal as possible. The Shapiro-Wilk tests show that all of the transformed variables except two are approximately normal. The measures of metabolite concentrations used for principal component analysis were Z-transformed to render them normally distributed on the same scale with mean of zero and standard deviation of one. Pre-post change in absolute metabolite levels were compared by repeated samples paired t-test. Baseline z-scores of metabolites were subjected to principal component analysis without rotation. PCA is an unsupervised analysis that aims to decrease the complexity of data by reducing variables to a smaller number of principal components . Each of the PCs were vectors of metabolite contributions. A direct oblimin rotation was used and 6 factors before the bend in the scree plot , and eigenvalues >1 were retained. Component scores for each participant were calculated with a standardized scoring coefficient. A PCA model with oblique rotation was tested to examine the factor correlation matrix. Since none of the factor correlations were over ± 0.32, indicating factor correlations are not driven by the data, we estimated a model with orthogonal rotation to reveal a simplified structure with interpretable factor loadings. To identify metabolite patterns of interest, bivariate Pearson-correlation analysis was performed between percent changes in metabolites and clinical biomarkers of CMR and with lipoprotein particle subfraction distributions separately. A generalized estimating equation procedure determined the significance of longitudinal changes in PC scores and their contributing metabolites using age and gender as co-variates. Wald Chi-square tests determined the significance of pairwise differences in treatment responses. Statistical analyses were performed using IBM SPSS Statistics version 25 and Rstudio version 1.1.456, and p-values <0.05 were considered statistically significant for the differences in traditional biomarkers. A Bonferroni correction for multiple comparisons adjusted the p-value set as significant to <0.002 for the correlational analyses with amino acid and ceramide metabolites.The study cohort was predominantly female, 61% Hispanic , 25% Nonhispanic Black, 8% Hispanic Black. and 6% Nonhispanic White, gutter berries with more Hispanic Not Black than Nonhispanic Black participants randomized by chance to the INT group . Full assessment of physical, behavioral, metabolic, and metabolomic status was conducted at baseline and study completion on the cohort of 36 participants . There was excellent attendance in both INT and CONT groups with all Teens and PAC participating in more than 78% of group sessions and 100% of baseline and follow-up assessment visits. There was considerable prevalence of obesity, dyslipidemia, inflammation and insulin resistance in all participants and mild hypertension in adult participants at baseline . Mean baseline BMI was over 30 mg/m2 in all participants, did not differ by sex, but was significantly higher in the CONT group relative to INT. The mean CRP was over 28.6 nmol/L in all participants but higher in Teen participants assigned to the CONT group than to the Teen INT group, and between the combined CONT and INT groups. Despite these random differences in BMI and CRP, central adiposity , blood pressure, lipids, insulin resistance and inflammation were similar in both groups, suggesting closely matched CMR profiles.

Although in population studies, Waist Circ is greater in males as compared with females, only one of the 18 PAC was male; while his Waist Circ fell below the adult female PAC mean, it was not the lowest recorded among PAC. The average Waist Circ for Teen males vs female Teens did not differ significantly so Waist Circ data are not reported by sex. Compliance with nutrient bar intake by self-report was 85.8 ± 11.1% and 86.7 ± 13.8% among INT group adults and Teens respectively.The quality of self-report diets assessed by Block food frequency questionnaire was poor in all participants at baseline . Modest but significant improvement in CONT PAC was evident in categories of saturated fat intake and added sugar but trends in the same direction did not reach significance in INT PAC or in Teens from either group. Average daily servings of dietary fruit , vegetables, not including potatoes , and fiber were comparably low in both CONT and INT groups and did not change in any age subgroup . Self-report activity increased in all participants in both groups . Weight was stable, even in the INT group despite the addition of 220 kcal in two daily nutrient bars. Good compliance with nutrient bar intake was suggested by a significant increase in plasma 25 hydroxy Vitamin D in the INT group PAC and Teens, but neither CONT subgroup. There were no other consistent effects of nutrient bar supplementation evident in traditional CMR biomarkers, nor were there significant within- or between group changes in any anthropometric measures in combined CONT and INT groups. Subgroup analysis of PAC and Teens separately showed some favorable changes in both CONT and INT groups. PAC CONT participants decreased Waist Circ . Among Teen participants, CRP trended lower in CONT but did not reach significance . Among Teens, systolic blood pressure decreased significantly by 7% in INT only, trending upward in CONT Teens with a significant between-group difference.Previous bar supplementation trials showed that chronic inflammation was associated with slower response in metabolic improvement. All participants, both Teens and PACs in this study, met criteria for obesity and had baseline CRP > 14.3 nmol/L.To test the hypothesis that subtle shifts in metabolism may precede changes in traditional biomarkers, a targeted analysis of plasma ceramides and amino acids was performed. The top six PCs extracted, each composed of distinct sets of linked metabolites, explained 59.3% of the total variance in the data set . PC1, which explained 16.7% of the variance, was composed exclusively of ceramides and ceramide-1-phophates; PC2 of amino acid metabolites; PC3 of sphingomyelins; PC4 of dihydroceramide species, glucosylceramide, and one specific ceramide subspecies Cer 18:1; PC5 of biomarkers of meat consumption and transsulfuration and polyamine metabolites ; and PC6 of amino acids in antioxidant defense .We next determined how increased physical activity by itself or with nutrient bar supplementation modulated these baseline metabolomic parameters. Analysis of PC change was performed with adjustments of potential covariates including age, gender, and baseline CRP. While there was no change in the plasma total ceramide pool, results showed significant divergence in several key ceramide species within PC1 between CONT and INT. Results show significant increases in C14:0 , C20:0 , C22:0 and C24:1 ceramides and a nearly significant increase in C16:0 in CONT. In contrast, C24:1 ceramide significantly decreased in the INT group by 18%; all other ceramides trended lower or remained unchanged . Analysis of group by time interactions showed significant between group differences in all of these C14:0, C16:0, C20:0, C22:0 and C24:1 ceramide species. Sphinganine and sphingosine are essential substrates for ceramide synthesis through de novo synthesis and salvage pathways. In CONT only, levels of sphinganine and sphingosine increased significantly by 59% and 50%, respectively resulting in significant pairwise differences between CONT and INT groups .Sphingosine-1-phosphate is a terminal breakdown product of ceramide and an important anti-inflammatory and vascular signaling lipid mediator associated with lipoproteins, particularly HDL, in plasma. As shown in Table 5, S1P levels increased differentially in both CONT and more so INT participants. The S1P increase in the INT group differed from the degree of change in CONT at p = 0.007. Table 6 lists amino acid metabolites with significant within group and between group changes.

There is no evidence of GLRaV-3 transovarial passage . While laboratory-based transmission studies have been informative, there is still a need for field-based transmission experiments to accurately estimate expected vector transmission efficiency and disease incubation time within the host. Information is lacking regarding how soon disease symptoms will appear after vector-borne infections are initiated, or how soon crop quality will be affected. The goal of our study was to obtain information about vector-borne transmission of GLRaV-3 and subsequent disease progression under commercial vineyard conditions. We performed a controlled GLRaV-3 transmission study into mature V. vinifera cv. Cabernet franc vines in Napa Valley, CA USA. We used first instars of the vector Pseudococcus maritimus , a mealybug species that is a common vineyard pest and native to North America . We performed a concurrent laboratory study, with identical experimental design, to compare pathogen transmission efficiency under controlled laboratory and field conditions. In the field study, dutch buckets system we estimated transmission efficiency and monitored time to GLRaV-3 detection via molecular diagnostics, appearance of symptoms, and effects of disease on berry quality.

Our findings are key to informing sound management practices with respect to understanding spread and progression of disease in a commercial vineyard. Furthermore, we provide a previously missing link between controlled laboratory studies and realistic vineyard conditions.Virus-infected dormant cuttings of V. vinifera cv Cabernet Sauvignon were used as source of GLRaV-3 in our transmission experiments. Foundation Plant Services at the University of Califtornia, Davis provided accession LV89-01 from their Virus Source Vineyard, which is known to be infected with genetic variant group III of GLRaV-3, Grapevine virus B , and Grapevine fleck virus . This accession was chosen because genetic variant group III of GLRaV-3 is common in Napa Valley . Plant cuttings were cut to three buds each, treated with RootBoost rooting hormone, planted in 1:1 vermiculite: perlite, and kept on a mist bench for 6 weeks, until a few leaves were produced and roots were approximately 2.5 cm long. Cuttings were then removed from the mist bench and transplanted to 10 cm pots with a growth medium consisting of 2:1:1 SuperSoil: perlite: sand , and kept in the greenhouse until used as virus sources in transmission experiments. GLRaV-3 infection was confirmed by molecular diagnostics prior to use for source material in transmission studies. Virus-free dormant V. vinifera cvPinot noir cuttings were also provided by Foundation Plant Services, collected during winter dormancy 2011, and propagated in the same manner as the virus-infected source cuttings. We used Ps. maritimus as the mealybug vector, which is difficult to maintain in insect colonies; therefore we relied on field collections for experimental inoculations. To obtain virus-free first instar Ps. maritimus mealybugs, third instar females were collected from a vineyard in Pope Valley, CA in May 2011, and allowed to mature and oviposit in the laboratory.

The third instar females were collected from underneath the bark of the trunks and cordons of mature grapevines and placed into gel capsules for transport to the laboratory. The mealybugs were immediately transferred to 100 mm petri dishes, each containing one piece of 70 cm Whatman filter paper. The mealybugs were kept in darkened conditions at 25 °C: 20 °C, 16: 8 h day: night temperatures- females were removed and discarded after oviposition. The filter papers containing ovisacs were pinned to the underside of the aforementioned infected source plants, which were then kept in a growth chamber until the first instar mealybug crawlers hatched. Approximately 72 h after hatching on the infected source plants, mealybugs were transferred to mature vines in the vineyard and to uninfected vines in the laboratory, for a 48 h inoculation access period. The timing of hatching led us to perform field inoculations on 18 July 2011, which coincided with the emergence of the new Ps. maritimus generation in Napa Valley. Twenty replicate source vines were propagated and used, with one to five recipient test vines inoculated per source plant in each inoculation experiment . All recipient test vines were treated with an insecticide upon completion of the inoculation access period.The experimental field inoculations were located in three rows of a vineyard block of V. vinifera cv. Cabernet Franc clone 01 grafted to 110R rootstock, obtained from Duarte Nursery and planted in Oakville, Napa Valley, CA in 1994. No vines in the experimental area were symptomatic for grapevine leaf roll disease prior to our experimental inoculations.

To confirm initial GLRaV-3-free status prior to inoculations, three petioles were collected from each experimental vine in July 2011 before inoculations were performed, for diagnostic testing . The block consisted of 8315 vines planted at 588 vines per hectare. Row spacing was 1.8 m, and vine spacing was 1.5 m, with a vertical shoot positioning trellis system and bilateral pruning. Row direction was northwest-southeast. Drip irrigation was provided using one 3.8 – L·h–1 emitter every 1.5 m. Experimental vines were spaced every third vine, and treatments were fully randomized. The three treatments included inoculations with no leaf cages, inoculations using mesh leaf cages, and negative controls for which no experimental manipulation was performed. Each treatment included 30 replicate vines, for a total of 90 experimental vines. The experiment comprised an area including 360 total vines, including the 90 experimental vines plus the spacer vines. The spacer vines were monitored periodically throughout the study for symptoms of grapevine leaf roll disease. A survey for any signs of mealybugs was performed in October 2012. On 11 October 2012, 15 months post inoculation, a commercial testing service collected and analyzed material from some vines that were symptomatic for grapevine leaf roll disease in the experiment and tested for a broad panel of known grape pathogens: GLRaV-1, GLRaV-2, GLRaV-2 strain Red Globe, GLRaV-3, GLRaV-4, GLRaV-4 strain 5, GLRaV-4 strain 6, GLRaV-4 strain 9, GLRaV-7, Syrah virus 1, Grapevine virus A, GVB, Grapevine virus D, Grapevine fanleaf virus, Xylella fastidiosa, GFkV, Rupestris stem pitting-associated virus, Rupestris stem pitting-associated virus strain Syrah, and Grapevine red blotch-associated virus. The vineyard block was not treated with insecticide prior to inoculations during the 2011 growing season. For inoculations, ten Ps. maritimus first instar insects were gently moved with a paintbrush from leaves of infected source plants onto the underside of one fully expanded mid-height leaf, located on a vertical cane growing from a middle spur on the south cordon of each grapevine. For the caged treatment, a cloth mesh cage was placed over the inoculated leaf and secured at the petiole using a twist tie. For the uncaged treatment, no covering was used on the inoculated vine. The experimental area was commercially treated with spirotetramat insecticide on 20 July 2011, after a 48 h inoculation access period. After inoculations the experimental area was managed following standard commercial practices.

Three months after inoculations, dutch buckets the petiole of the inoculated leaf was collected on 14 October 2011 for diagnostic testing. In the instance where that petiole had fallen off the vine or could not be found, a petiole near the inoculated leaf was collected; inoculated petioles were missing from 9 of 60 inoculated vines. Immediately following the first appearance of symptoms in 2012 and 2013, petioles were collected from each experimental vine and tested for presence of GLRaV-3. Petioles were collected from each experimental vine in September 2014, and tested for the presence of GLRaV-3, GVB, and GFkV. On each sampling date, three petioles were collected from each vine and pooled for diagnostic testing. If a vine had symptomatic leaves at the time of sample collection, symptomatic leaves were preferentially collected over asymptomatic leaves. During each growing season in 2011 through 2014 , experimental vines were surveyed regularly for visible leafroll disease symptoms, beginning immediately after inoculations. On each survey date vines were marked as either asymptomatic or symptomatic, with surveys beginning in May and continuing through October. Shortly after symptoms first emerged in 2012, a detailed symptom survey of each symptomatic vine was performed to determine possible variation in disease symptom severity among vines and if there was an association between location of inoculation and initial appearance of symptoms within vines. For this survey, the position of each spur and the number of symptomatic and asymptomatic leaves on each spur were recorded. In Year Two, berry quality of all vines was measured three times during the weeks immediately preceding commercial harvest. Degrees Brix , pH, and titratable acidity were measured on 31 August, 21 September, and 3 October 2012, and harvest was 4 October 2012. In Year Three, berry quality of a randomly selected subset of 30 vines was measured on 28 August and 14 September, and harvest was 14 September 2013. The 30 vines were evenly divided between uninfected negative controls, uninfected and infected vines from the caged inoculation treatment, and uninfected and infected vines from the uncaged inoculated treatment. For berry quality analysis, on each sampling date approximately 200 berries were collected from each vine to minimize variance in measurements . Within each grapevine, berries were collected from the top, middle, and bottom of each harvestable cluster of grapes and pooled for laboratory analysis. All samples were processed by Constellation Laboratories in Califtornia, USA. Total soluble solids as °Brix were measured using an Atago refractometer, and pH was measured using an Orion pH meter. Titratable acidity of the juice was measured via direct titration with 0.1 N NaOH, using phenolphthalein as an indicator.To compare field inoculations in an established commercial vineyard with controlled laboratory studies, a laboratory experiment including the same treatments was performed, using the same collection of Ps. maritimus and same known infected source plants. Ps. maritimus first instars were moved from known infected source plants to uninfected recipient test plants on 15 July 2011 and allowed a 48 h inoculation access period, after which plants were treated with a contact insecticide. Mealybugs on half of the recipient test plants were caged on the underside of one leaf using clip cages, as described by Tsai et al. , and mealybugs remained uncaged on the other half of the recipient test plants. Thirty replicate recipient test plants were used for each treatment, caged and uncaged, for a total of 60 recipient test plants. Additionally, a replicate of 30 uninoculated plants was kept in the greenhouse following inoculations to assure that all GLRaV-3 infections were due to our experimental inoculations. Following inoculations, plants were kept in a greenhouse with 16:8 light: dark using supplemental lighting as needed, and 25 °C: 22 °C day: night temperatures, with regular fertilizer and insecticide treatments. Because disease symptoms under greenhouse conditions are not reliable for diagnosis of GLRaV-3, we used molecular testing to confirm infection with GLRaV-3. Four months after inoculations, at which time new GLRaV-3 infections can be reliably detected using molecular diagnostics, three petioles per inoculated test plant were collected and stored at −80 °C until testing for GLRaV-3 .To test whether the newly infected field vines could be a source of GLRaV-3 one season after mealybug inoculations, a transmission experiment was performed in the laboratory from cuttings of these newly infected fieldvines. Ps. maritimus were not used because of the abovementioned difficulty in obtaining virus-free first instars for transmission experiments. Instead we used first instars of Planococcus ficus, which are easily maintained in colonies and therefore can be ready for use in transmission studies at any time. Furthermore, Pl. ficus is a known vector of GLRaV-3 . Field cuttings were collected on 4 October 2012 and the stem bases were placed in flasks of water. First instar Pl. ficus were allowed a 24 h acquisition access period on the field cuttings, then transferred to the underside of a leaf of virus- free V. vinifera cv. Pinot noir recipient test vines; ten insects per recipient test vine were confined using a leaf cage for a 24 h inoculation access period. Following inoculations, plants were treated with a contact insecticide and then kept in a greenhouse for four months until petiole sample collection for diagnostic detection of GLRaV-3. For this experiment, a randomly selected subset of experimental field vines of each treatment was tested as a potential GLRaV-3 source. In total, nine symptomatic vines were tested; five from the caged inoculation treatment and four from the open inoculation treatment, and seven recipient test vines were inoculated in the laboratory from each symptomatic field vine. One of these 63 recipient test vines died before petiole sample collection to test for infection with GLRaV-3.

Our modification accounts for the effect of temperature on the survival and fertility of D. suzukii in calculating population growth of the organism. Typically, researchers have introduced elements of environmental stochasticity to matrix models to study environmental effects on population trajectories. However, our approach relies on temperature-dependent estimations of age specific fecundities and survival that are determined by models fit to lift table data generated for multiple temperatures. Our environmentally dependent matrix model is unique in that it does not rely on simulation of environmental effects on populations, but the matrix itself is recalculated at each iteration in direct response to environmental input. Model predictions were run under environmental conditions from different regions to illustrate variation between and within study sites in different years. These simulations make important predictions about age structure and population trends that have implications for pest management both in a broad sense and with regional specificity. This modeling tool may improve current management practices by predicting pest pressure independent of trap catches or samples of infested fruit. We also see potential applications of this model for research in other fields of study and for broadening the understanding of how pests interact with the environment.

The population projection model was written in the open source statistical environment R version 3.0.2. The model calculated the matrix based on mean temperature input. Briefly, nft hydroponic the matrix calculations were based on age-specific regressions of temperature-dependent population parameters as highlighted by Tochen et al.. Whereas immature lift stages of D. suzukii may experience different environmental conditions than adults because these lift stages are completed within the fruit, in this study, ambient air temperatures were used to predict population dynamics for all lift stages. To return age-specific population vectors for 50 age-classes of D. suzukii for each test case, a vector of mean daily temperatures for each site was input into the R statistical interface. The biofix, or the point where the model began in the spring, was determined using methods described in Tochen et al.. Biofix essentially described the earliest point in the season when the temperature allows the population to increase. Calculations for population estimates were initiated on the biofix date of 2 February in Parlier and 1 April in Wilmington and Salem . In Pergine and Sant’Orsola, estimates were initiated on 6 April . The population matrices were initiated with 100 flies in the population vector for 41–50 day-old females based on the assumption that females of this age group represent flies that would be emerging from diapause in spring. The log transformed sum of D. suzukii from all lifte stages for each day represented the total population estimate except where age distributions are considered. For daily age distribution of D. suzukii from Parlier, Salem and Wilmington during 2013, 1–3 day-old D. suzukii were classified as eggs, 4–7 day-olds were larvae, 8–9 day-olds were pupae, and 10–50 day-olds were classified as adults.

Among the most important assumptions of the model are that populations of D. suzukii would not be limited by host availability, are not density dependent, do not exhibit Allee effects, and that response to current temperature is not dependent on previous temperature exposure.Seasonal weekly trap catches of D. suzukii were recorded in all study sites, except Riva del Garda, but model estimations for this location was included because the climate here is much different from the other locations studied in Italy. Trap counts were pooled data from commercial blueberry fields in Wilmington ; unsprayed apricot, blackberry, blueberry, cherry, peach, and citrus orchards in Parlier ; commercial blueberry fields and surrounding blackberry vegetation in Salem ; strawberry, blackberry, cherry and blueberry fields in Pergine ; and unsprayed strawberry and raspberry fields in Sant’Orsola . In Wilmington and Salem, traps were made of clear plastic cups, ca. 1 liter in volume each. Each trap had 6–15 entrance holes 4.5– 9 mm in diameter. Trap baits in Wilmington consisted of a yeast and sugar water mixture containing 6 g yeast and 40 g sugar dissolved in 710 ml water. In Salem, traps were baited with 100– 200 ml natural apple cider vinegar and 1–3 ml unscented liquid soap to break water surface tension. In Parlier the traps were made to the specifications of the ‘‘Haviland Trap’’ design for D. suzukii monitoring. A 750-ml plastic container served as the basin for each trap. A 7.5-cm diameter hole was cut in the lid, over which a piece of 0.6-cm wire mesh was attached. Each trap was covered with a Pherocon trap cover , which had a built-in wire hanger. Each trap was filled with 250– 300 ml of apple cider vinegar with 15 ml of unscented soap added as a surfactant to each container of vinegar. In Trentino the containers were 1000-ml graduated white polyethylene bottles filled with 200 ml apple cider vinegar . All traps were placed near the fruiting level of host plants or on stable surfaces in shaded areas and were checked weekly.

The contents of each trap were collected into a separate container that was taken to the laboratory for processing, and at the same time, the traps were refilled with fresh apple cider vinegar and unscented soap, as described above, in the field. The liquid and contents from each trap sample were strained in the laboratory and the numbers of adult SWD collected were recorded by gender. All data from traps were analyzed to display mean weekly D. suzukii per trap for each of the regions. Mean daily temperatures for all seasons are presented together with trap catches.In Wilmington, winter and late dormant mean temperatures were never below 0uC and reached 20uC multiple times prior to 31 April . At this site, mean high summer temperatures exceeded 25uC multiple times during July, and mean low temperatures were below 10uC during November. In Parlier, winter and late dormant temperatures were never below 0uC and were higher than observed in both Salem and Wilmington during this period . Here temperatures were above 25uC multiple times prior to 31 April. Mean daily temperatures were consistently above 25uC during June–September, and daily means in Parlier sometimes exceeded 33uC. In Parlier, temperatures dropped to below 10uC during November. In Salem, early-season temperatures were warmer during January and February of 2012, compared to 2013 . Temperatures observed from March through May were slightly higher during 2013 compared to 2012. In Salem, winter and late dormant temperatures were never below 0uC or above 15.3uC until 31 April 2013 . Daily mean temperatures gradually increased to 25uC during July, after which daily mean temperatures dropped to below 10uC during November. For the Italian sites Riva del Garda had higher late dormant and fall temperatures than Pergine and then by Sant’Orsola . Summer temperatures were similarly ranked higher in Riva del Garda, followed by Pergine and then Sant’Orsola during 2013. In Pergine during winter 2012 , late dormant temperatures were frequently below 0uC and were as high as 20uC before 31 April. Fluctuations of temperature were more pronounced in 2012 in Pergine, compared to 2013 . Very low temperatures were recorded in Pergine from 3–12 February 2012, followed by relatively warm temperatures from 24 February to 13 March. Temperatures were also comparatively low from 9–11 April 2012. In Pergine, daily mean temperatures increased to 25uC during July, after which they dropped to below 10uC during November. Mean temperatures were well below 0uC during December.Population estimates using temperature data indicate that D. suzukii populations are able to increase to high levels in all of the studied locations . When comparing early-season population estimates between Wilmington, Parlier, and Salem , hydroponic gutter the population estimates were highest in 2013 in Wilmington followed by Parlier and then Salem. However, the population estimate for Salem surpassed Wilmington by 15 June and surpassed Parlier on 16 July, as Salem population estimates continued to climb while the latter sites experienced declining populations after reaching the first peak of their bimodal distributions. In Parlier, the early-season population peaked on 16 June, subsequently decreasing to a low on 10 September before increasing to a second population peak on 9 November, then decreasing again as winter progressed.

In Wilmington, the population curve peaked on 21 June, then the population curve declined slightly for an extended period, followed by a second period of population increase beginning on 19 September to a population peak in November. In Salem, populations consistently increased from 25 April to a peak on 22 October, followed by a steep decrease. When comparing population estimates between seasons for the initial harvest period of early- to mid-season blueberries in Salem , the majority of model outputs for this period estimated greater populations for 2013. When comparing populations along the elevation gradient of the three Italian sites, higher early-season populations were predicted at the lowest elevation Riva del Garda, followed by Pergine and then Sant’Orsola . In Pergine, greater population numbers were estimated for the majority of the growing season during 2013 compared to 2012 . In all model predictions, immature lift stages comprised by far the majority of the population, except at the beginning or end of the season when adults tended to dominate . One exception was Wilmington, where temperatures remained favorable for reproduction into the late fall so that immature stages remained a majority of the population . In Salem, fall temperatures initially caused cessation of reproduction, leaving a majority of adults, but December temperatures allowed for some reproductive activity to occur . In the early spring, a higher relative percentage of adults occurred due to the overwintering adults that were initiating their first reproduction. In part, this was an artifact of initiating the model with only older adult females. In the fall, environmental conditions became unfavorable for reproduction but may not have had strong effects on adult survival. Overall, no populations reached a completely stable age structure, but the highest relative stability for each site occurred in the middle of the season. Stability of age structure was the highest in Wilmington, followed by Parlier and finally Salem, which had a high degree of instability . Demarcation of distinct generations was very clear for the first part of the season in Parlier and Salem , but during the mid season at these sites and in Wilmington , it was very difficult to distinguish individual generations to distinguish complete generations from partial generations.In Wilmington, D. suzukii counts were first recorded on 5 May 2013 at one fly per trap with an erratic increase to a peak in numbers at 26 flies per trap on 26 July . After this period, the trap numbers gradually decreased to six flies per trap until 4 December, at which point D. suzukii trapping was discontinued. In Parlier, two population peaks were found during the crop season, one during the early part of the season, followed by a long mid-summer period without fly captures, and a second peak during the latter portion of the season . Adult D. suzukii were first caught on 19 March 2013 at one fly per trap and increased to a high of six flies per trap on 16 May, after which they decreased to zero on 27 July. The trap numbers remained at this level until 19 September, after which numbers continued to increase into December. In Salem and Wilmington only one population peak was observed during the summer period . During 2012 in Salem , D. suzukii trap counts consistently increased starting on 5 July from one fly per trap per week to a maximum average of 17 flies per trap on 6September. During 2013 in Salem , the first D. suzukii trap counts were observed on 30 May at an average of three flies per trap per week and gradually increased until 10 September, when a maximum of 27 flies per trap was observed. The first trap counts during 2013 were therefore consistently recorded four weeks before those found in 2012 and higher levels of flies were found in traps during 2013 in Salem. In Pergine and Sant’Orsola, fly counts were first observed 23 June 2013 , and on 7 July 2013 in respectively. In Italy, one population peak was visible each year for Pergine and Sant’Orsola. In 2012 in Pergine, the first flies were trapped on 25 July, approximately four weeks before those caught during 2013.

Direct competition occurs only among the betilids species, while indirect competition occurs with P. coffea. Gutierrez et al. considered symmetric competition among the betilids, but new data enables parametrization of asymmetric competition in dyadic contests the success rate of which depends on their biology . The different success rates are included in the model via the parameter ı , which weights the proportion of contest that are successful for the dif-ferent combinations of species. No information is available on rare triadic interactions, and hence is not considered in our analysis. When a second species of a betilid parasitoid enters a berry previously colonized by another parasitoid species, it attacks the immature stages of both CBB and the first parasitoid. Facultative oviposition and host-feeding interactions between betilid species are summarized in Table 4 .Simulations for Colombia had a five year time horizon corresponding to the normal crop cycle . In Brazil, the crop cycle is four years. In Colombia, two coffee harvests occur per year, a main one in September-November and a minor harvest in April-June . At Londrina PR , plastic flower pots only one coffee harvest occurs per year that may occur during June through September .

The ratio of parasitoid released was 1 per 1 CBB infested berry. In Colombia, the first berries colonized by CBB females appear around 5300 dd after planting , and at 7500 dd in Brazil. The times of berries first appear are reference times for parasitoid release. In Colombia, an initial release of 11 adults/plant of each betilid species occurs at 6790 dd and corresponds to a ratio of 1:1 parasitoid adults to CBB infested berries. For P. coffea, the initial release was 5 adults/plant corresponding to a ratio of 1:1 parasitoid females to CBB infested berries at time 5678 dd. For Brazil, the initial release of each of the betilid parasitoids at 8500 dd is 4 adults/plant corresponding to a 1:1 ratio of adult females of each betilid species to CBB infested berries. For P. coffea, 6 adults were released at 7500 dd for a 1:1 ratio of parasitoids to CBB infested berries. Single and multiple parasitoid releases during the crop cycle were also evaluated. Two strategies for multiple releases were evaluated: the first consisted of one release at the start of each coffee production cycle when CBB attack begins and the second in had releases at the start and end of each production cycle. The release rates were one female parasitoid per infested berry. The season long damage reduction for each scenario is computed as the ratio of CBB infested berries with control/CBB infested berries without control, while the proportion of parasitoid colonization rate by each species was computed as the ratio of CBB infested berries with parasitoid/total CBB infested berries. A constant immigration rate of 0.25 CBB females per day and plant was used in the model, while no immigration was assumed for the parasitoids. Dyadic contests, facultative host-feeding and facultative hyperparasiThism as appropriate for each species were examined for all combinations of parasitoid species interactions .

The effects of interspecific competition were analyzed using the number of adult parasitoids of each species produced per plant as affected by the presence of competing parasitoid species.The effects of weather at five locations on the coffee agroecosystem were analyzed for the 24 = 16 combinations of the four parasitoids including the null set. The different combinations in the model were characterized using Boolean presence , absence values. The efficacy of each parasitoid species on CBB control was further evaluated using permutations of the years of weather data from the five locations obtained using the allPerms procedure of the “permute” package in the statistical software R . For Colombia, this resulted in a random subset of 24 combinations of yearly weather data and 16 combinations of parasitoids . A similar procedure was used to generate 128 scenarios for Brazil.Negative binomial mixed regression models were fit to simulation outputs. This method accounts for over dispersion and skewness in the data, saThisfies the assumptions of the parametric analysis, and allows interpretation of the stochastic effect of the combinations of years of weather obtained by the shuffling procedure. In the analysis, the Boolean variables are the independent variables, while the cumulative number of CBB infested berries·year−1 in the simulation runs are the dependent variables. The effects of species-specific biology such as facultative host-feeding, facultative hyperparasiThism and dyadic contests were critical underlying factors affecting the output of the model .

The simulation runs were used to estimate the effects of parasitoid species on CBB infestation levels and oneachother.Akaike’s information criteria was used to select the final model for the different combinations of parasitoids, and chi-square goodness of fittests were used to assess the significance level.The dynamics of CBB infested berries in the absence of the parasitoids are depicted in Fig. 2a and predict CBB infestation of berries of 64.67%. Because parasitoid releases at the beginning and at the end of each harvest period for each species acting alone gave only slightly better control than the single release strategy , only the results for single releases are illustrated in Fig. 2. A single release of C. stephanoderis at 10 CBB infested berries/plant resulted in a 21.06% reduction of CBB infested berries on the plant and an 11.35% reduction in infestetation in shed berries on the ground. Compared to C. stephanoderis, a single release of C. hyalinipennis resulted in a 16.93% reduction in infestation on the plant and 8.23% reduction in infestation in shed berries, whilst a release of P. nasuta yielded 16.83% and 7.59% reductions respectively. In contrast, a single release of the eulophid P. coffea resulted in a 47.38% reduction in CBB infested berries on the plant and a 46.85% reduction in infestation in shed berries. Operating singly, the order of effectiveness of the parasitoids is P. coffea >> C. stephanoderis > C. hyalinipennis = P. nasuta , but none of the parasitoids provided high levels of control. Simulation of CBB infested berries available for attack by the betilid species shows asynchrony between seeking adult parasitoids and the low availability of CBB infested berries . The low level of control by single releases of the betilids is the consequence of the lag between the peaks of colonization of CBB infested berries attacked by the betilids and the late emergence of searching betilid females at the end of the ripening season . of Regressions of CBB infested berries·year−1 on the presence the parasitoid species under multiple climatic conditions shows that only P. coffea has a significant impact in reducing CBB infestation levels , a level of control that is clearly insufficient.From a human perspective, control of coffee berry borer using effective augmentative and/or classical biological control is desirable, but this outcome appears not to be possible in Central and South America using the three commonly introduced parasitoids . Of these parasitoids, only P. nasuta established in Colombia , and only C. stephanoderis and P. nasuta established in Brazil . A fourth parasitoid, C. hyalinipennis was considered for introductions, but laboratory studies revealed it is a facultative predator and hyperparasite on immature stages of the other betilids and conspecifics, plastic garden container it super parasiThism of CBB larvae, and it does not attack CBB adults . Our simulation results confirm the impact of these detrimental effects.We sought to determine why the establishment of the parasitoid species in the face of high CBB infestations in Colombia and Brazil has been difficult, and why no combination of the parasitoids shows promise for effective control of CBB. We first review the simulation results and then relate them to ecological theory. In sharp contrast to what is observed in the field, we assumed that all of the parasitoids established in the field and are attacking CBB and interacting with each other. This discrepancy with the facts suggests deficiencies in our knowledge of the biology of the species, and the omission in the model of the limiting effects of harvesting, crop management and cultural controls, and alternate hosts. For these reasons, our analysis of the parasitoids for control of CBB must be viewed as heuristic.

Earlier simulation results by Gutierrez et al. and here suggest that only the eulophid adult endo-parasitoid can reduce CBB populations significantly but not sufficiently. Unlike the betilids, P. coffea attacks multiple CBB females as they are boring into coffee berries and effectively stops CBB reproduction. Furthermore, P. coffea does not host-feed on siblings, allowing rapid growth of its populations during the berry-ripening period. Studies with P. coffea in Costa Rica and Mexico using sleeve and field cages support our simulation results. In Costa Rica, Espinoza et al. released a 1:10 ratio of P. coffea adults to CBB infested berries in entomological sleeves and obtained 95% parasitization of CBB adults and a 3 − 5.6 fold decrease in berry damage. In a similar study in México, Infante et al. obtained P. coffea parasiThism rates of 79%. Unfortunately, cage results cannot be compared with open field releases such as those of Castillo et al. who obtained a very low progeny from the original number of female adults liberated. In a two year survey of CBB parasitoids in Western Kenya, only 17 P. coffea adults were reared from 69,500 CBB infestedberries . These differences in the field studies suggest that crop management, harvest intensity, weather and other unexplored factors must play large roles that will be explored in a forthcoming report.Gutierrez et al. found that the betilids released singly or in combination assuming high immigration and search rates gave poor control of CBB. They attributed the result to a low numerical parasitoid response relative to CBB as each betilid female colonizes only one berry and only partial mortality of the available hosts may occur. Our results confirm this interpretation. Further, the oviposition rate of betilids estimated from field data ranged from 0.055 to 0.076 eggs·female−1·dd−1 which is about six fold lower than the oviposition rate of 0.348 eggs CBB female−1·dd−1 . In addition, host-feeding by betilid adults on CBB eggs and larvae is common and reduces the future availability of preferred stages for parasiThism. Under laboratory conditions, C. stephanoderis females fed honey had 80% greater longevity and decreased host feeding, and this practice has been proposed to improve the survival in the field after release and to improve control of CBB, especially when preys are scarce . In Colombia, high densities of betilid females emerge at the end ofthe berry ripening seasons when CBB infested berries are in short supply. Thus, despite predicted high parasitization rates, a bottleneck occurs between harvests that reduces adult betilid densities to levels that are slow to rebuild in the face of rapidly increasing CBB populations. This result was verified by Benassi in the states of Espiritu Santo and São Paulo, Brazil who observed that low densities of wasps at the beginning of the harvest period required several months to increase significantly. Benavides et al. suggested that parasitoid releases should be made during the middle of the ripening season, but high CBB damage rates normally occur by this time.Whatever strategy a species evolves, it must assure its continuity in time, and hence its traits must assure “adaptedness” to the biotic and abiotic conditions to minimize the likelihood of extinction . Further, Gutierrez and Regev proposed that evolved strategies may have little to do with maximizing the commonness of the species in the environments. Rather, adapted individuals and the species they comprise must via their genetics, take a long-term view of their resource management problem, and that tradeoffs are based on sufficiency or constrained optimization behavior rather than maximization rules. On the theoretical r-K continuum , coffee and CBB have r-selected strategies, while the parasitoids have intermediate ones. The well-documented biology of coffee, CBB and its parasitoids suggests that poor control of CBB is not unexpected because coffee is a long-lived shrub that produces high numbers of fruit, and few seeds need survive CBB attack to provide replacements . Furthermore, CBB is polyphagous in its home range in Africa with coffee being but one of its hosts . Similarly, the linkages of the parasitoids to CBB are not particularly tight because the parasitoids also have other hosts and CBB and the parasitoids spread the risk of extinction through polyphagy . As a result, little by way of regulation of CBB occurs resulting in high infestation levels in coffee in the Americas and in its native range in Africa.