We are starting a new set of insect rearing courses, beginning with Course 1: Insect Diet Science. The course consists of 20 hours of online (Zoom) instruction from Professor Allen Carson Cohen, author of Insect Diets: Science and Technology and more recently Design, Operation, and Control of Insect Rearing Systems. The course requires a $250 registration fee, giving the participant access to the lectures and discussion groups and the reference materials specific to each topic. Classes are given on Tuesdays and Thursdays from 1:00 to 3:30 pm Eastern Time.
For more information or inquiries, you are encouraged to contact Professor Cohen at email@example.com
In the previous post, I mentioned the concept of granulometry as part of the approach to understanding matrix relationships–in this case, the particle-particle interactions.
The above figure is a representation of the image in the previous discussion where the following modifications were made to the original figure: 1) the color pattern was changed in an image processing step performed in Microsoft PowerPoint. The technique changes colors and adds emphasis and contrast to the different portions of the image; 2) portions of the figure that contain wheat germ (WG) and yeast concentrations (YC) were marked, and some of the suggested interactions were depicted with the arrows; 3) the insets represent two different kinds of granulometric analyses where the coarse diet solids (wheat germ and torula yeast) on the lower right and fine particles on the lower left were separated with a set of mechanised sifting screens. Particles within each size range were weighed and the size distributions graphed with the bar graphs. The bar graphs represent these particle sizes: > 2000 microns; 1000-2000 microns; 500-1000 microns; 250-500 microns; 125-250 microns; 65-125 microns; and < 65 microns. The different sizes of particles between the coarse and fine particles were produced by using mixtures of wheat germ (20 g) and torula yeast (10 g) of non-milled solids vs. the same proportions of the solids that had been run through a cutting mill.
In association with the differences in particle size, the diet made from the coarse particles had a firmness (gel strength) that was about 1.5 times as great as that of the gel made with fine particles. This is an example of the kinds of effects that matrix interactions can influence in various artificial diets. Part of our current research in the Insect Rearing Education and Research Program at NCSU is to discover the various kinds of matrix characteristics that arise from use of various diet materials and diet-processes. A central line of inquiry deals with the physicochemical characteristics of different factors in diet production.
In yesterday’s post, I discussed the matrix concept in insect diets. I used an example of a visual treatment of a diet matrix using an image of a stained wheat germ-based diet viewed and photographed under about 20 x magnification in a video microscopy system. The basic image looked like this:
In the above images, the lower image shows a 1 x 3 cm section of diet with hornworms consuming the diet. The neonates (newly-hatched 1st instar larvae) depend upon finding all the nutrients they require within a small area of the diet where all lipids, protein, carbohydrates, minerals, and vitamins must be present in adequate amounts to support healthy growth. The upper image shows the granular nature of the diet whose particles (which are suspended in an agar or carrageenan framework) have an organization that can be thought of as conforming to a network, matrix relationship. This relationship (the granulometery) contributes to the potential for structural integrity, resulting in diet consistency and texture. There is also a great deal of potential chemical interaction such as lipid-binding by lipoproteins, carbohydrate-protein interactions, enzymatic and oxidative reactions, diffusion of solutes or nano-particles that are involved in the diet’s taste/texture qualities (palatability), the availability of nutrients within the reach of the insect’s mouthparts; then upon ingestion, the particles and solutes must be available for digestion and absorption. Finally, the arrangement of the particles and other diet components must lend itself to the diet’r retention of palatability, nutritional value, and bioavailability in a framework that we call stability. All the physicochemical characteristics and interactions constitute the diet’s MATRIX.
In these images, there is an implicit matrix organization that dictates or commands the nature of the diets in question. To better understand this matrix/organization feature, we can examine the following diagram:
In the above diagram (partly explained in the image caption), the various levels of organization are shown from macro- to nano levels. The inset on the right-central part of the diagram is a fluorescent micrograph of the diet that was stained with the lipid stain, Nile Red, which shows the lipids in a 20 micron particle of wheat germ. Other fragments in this insect (against the black background) are brightly-coloured carbohydrates, which stain blueish with Nile Red. The upper central portion is a diagram of a large lipoprotein molecule of about 25 nm x 5 nm, and on the left central portion a sterol molecule is depicted as a less than 1 nm structure. The lipoprotein molecule contains hydrophobic pockets (the greenish structures on the left and right ends of the molecular structure). It is the hydrophobic pockets in lipoproteins that bind with and carry lipids (= lipophorins). Throughout this diagram, it is evident that there are micro-relationships on the physicochemical level of organization, and these relationships lead to degrees of stability, nutrient bioavailability, and taste (palatability). An important part of this relationship is in the interactions between particles and liquid interfaces, and we will further explore these in relationships that we study with granulometry.
At the centre of insect rearing systems are diets, usually artificial diets. These diets are composed of multiple components such as base nutrients and functional components. Base nutrients are usually complex nutrient “packages” such as wheat germ, soy flour, chicken eggs, corn meal, meat products, yeast products, rice flour, wheat bran, oat bran, and dozens of other foods that contain proteins, carbohydrates, lipids, minerals, vitamins, and various biochemical components such as flavonoids, phenolics, etc. Many of the base nutrients common to insect diets are nearly complete nutrient packages that are supplemented by other components to complete the nutritional and phagostimulatory (feeding stimulation) of the artificial diet. If a component such as wheat germ is found to have a deficiency in carbohydrate or lipid, for example, sucrose or starch and linseed oil or cholesterol may be added to complete the nutritional or phagostimulatory needs of the insects. Salt (mineral) and vitamin mixtures are also often added to complete the nutritional package.
Other artificial diet components that help give the diet consistency and stability are added to meet our target insect’s specific feeding characteristics. For example, many insects that feed on plant tissues do well with gelled diets, so we use gelling agents such as agar, carrageenan, or other substances that bind water and diet components in such a way that the consistency of plant leaves, stems, roots, or fruits is mimicked enough that the insect accepts the diet as a suitable plant-part substitute. We also add materials that reduce microbial spoilage or oxidative deterioration (anti-microbial agents and antioxidants) to stabilize the diet. While the topic of nutritional and functional components of diets deserves much more attention, for the present discussion, let us say that the diets that are designed to provide complete nutrition, palatability, bioavailability, and stability are the diets that we deem “successful” (See Cohen 2004 and 2015 for further discussion of the nature of successful diets).
However, one of the facts of life that became apparent to me decades ago is that many diets that people had tried to develop failed to support complete development of successive generations of target insects. Even though ALL the seemingly necessary components were present, often, our diets failed!
I came to realize that “the whole is greater than the sum of its parts,” in diet development research. I found that I could take a good diet as as living or frozen prey of a predator, break down the components, and then reassemble them and I would NOT have a diet that worked as well as the original food. I found further that it was not just a question of the food being alive that related to the food’s success. It was something in the organization of the food’s components that conferred (in part anyway) success of the diet. The organization is another way of discussing the matrix nature of the diet and its constituent components. I offer an image of a diet that shows how components function at various levels of organization (gross macro-, macro-, micro-, and nano-levels of organization.)
I will further discuss this diagram and its MATRIX nature in further discussions on these pages. I also point out that in my classes, I try to provide an intense and. detailed documentation and process of inquiry into the matrix nature of diets that work=successful diets.
A problem that I have found in the insect rearing community is that we rearing specialists tend to view our domain as a strictly applied discipline. I try to counter this trend in my rearing courses. The problem is that as rearing specialists, we have a very narrow purpose in our job description: producing the target insect. The complexities of this job are so great that it’s easy to see why a busy rearing scientist would not find time to scan the literature looking for research findings that MAY be applicable to our target insect.
What I try to do in my courses is to make constant efforts to expand my own base of understanding and knowledge, and I further try to provide a base of information on the issues that give the students an appreciation for how seemingly disparate information may be of great value in understanding their insects in a rearing context.
One example of this is the recent discussions that I have posted on oxygen and carbon dioxide in our rearing systems. My experience of working with wax worms (larval Galleria mellonella) called my attention to the issue about whether or not the larvae were able to get sufficient O2 (and to void the CO2) that was produced by these crowded larvae (see Figure 1). Early in my waxworm rearing experiences, I learned that the larvae tend to aggregate and display high levels of activity, sometimes just pulsing back and forth in their silk tunnels. I saw this phenomenon in many different containers that I tested to optimise the rearing cages for my rearing system. From my observations, I became convinced that the larvae spend a great deal of their time and their silk-making resources in a quest for gas exchange.
This very applied question led me to investigate the gas exchange issues with G. mellonella larvae, and one of my current research topics is the determination of respiratory dynamics with various diets (low vs. high fat/ low vs. high carbohydrate diet mixtures). These studies have further led me to measure the O2 and CO2 in rearing containers where the waxworms crowd themselves in tight aggregations with high density populations. Naturally, the discovery that the waxworms were chronically under low O2 and high CO2 conditions led me to ask the questions about O2 and CO2 dynamics of other reared insects, especially the gregarious ones that we keep at high densities. This line of thinking led me to search the literature for various publications on the effects of O2 and CO2 stress (low oxygen and high carbon dioxide conditions). It was during this line of inquiry that I found the paper that I have been discussing from VandenBrooks et al. 2018 where the authors demonstrated the effects of hypoxia manifesting itself with enrichment of tracheoles and mitochondria. This very basic science discovery may have far-reaching implications for insect rearing where the practical (applied) issue of quality control of our insects can be directly related to availability of O2 and dispensing of CO2.
More about these relationships in further posts, but let me re-emphasize the importance of being flexible minded about what is “mere basic science” vs. what has very practical implications in our rearing systems.
In all my courses, I try to emphasise how we can use critical thinking and basic science to better understand all the components in our rearing systems and how these components interact to make the systems function. I designed the diagram to be complex-looking to emphasise the many, many intricacies of rearing systems, especially the many kinds of interactions between the numerous “moving parts.”
In this diagram, I tried to represent how we can zoom-in on facets of the rearing system so that we can understand them in a piecemeal fashion, which leads to an overall understanding. Philosophers of science might call this going from the specific to the general, which is characteristic of inductive reasoning. My approach to rearing systems is to explore relationships inductively to arrive at generalisations, and then use the generalisations to help us better understand and predict the specifics (= deductive thinking). This kind of thinking is not new to me; it was approached by such famous scientists as Claude Bernard (1813-1878) and the person who suggested the concept milieu interieur, which is the basis of our concept of homeostasis.
In the diagram in Figure 1, a relationship between metabolic rate–oxygen–tracheal diameter is shown. This concept is discussed in reference to a paper that I cited from VandenBrooks et al. 2018 who showed that in response to lower than normal atmospheric oxygen levels, Drosophila melanogaster developed larger, more complex tracheal systems (tracheole branching, diameter, number) and more mitochondria while in higher than normal O2 atmospheres, the insects showed the opposite trends. In the post where I first raised this issue, I suggested that this phenomenon could be taking place in other insects, and I further suggested that under rearing conditions where the O2 levels were not as low as the experimental values used by VandenBrooks et al., the same types of changes in respiratory organs and organelles may follow the same trend, but possibly to a lesser extent. I further provided data from some of my rearing observations where I showed that indeed, under commonly found rearing conditions, insects displayed lowered O2 and elevated CO2, though my observations did not find such low levels as those imposed by the VandenBrooks team.
This is where my point about critical thinking comes in. A published study demonstrated that a series of very dramatic changes took place in one insect species’ respiratory system morphology and ultrastructure. Does this mean that such changes take place in other insects (such as my wax worms or somewhat crowded painted lady butterflies)? The finding with D. melanogaster does not establish that the types of changes discussed here take place in my subjects or in the other insects that are routinely reared. Also, the extent of the hypoxia (low oxygen tension) does not necessarily extend to lesser hypoxic conditions. An important part of the critical thinking that I am encouraging in my courses is that rearing personnel are well-served to take what we know about insect physiology, biochemistry, genetics, ecology, microbial relations, etc. and consider how such basic science knowledge MAY apply to their insects in their rearing systems.
This approach encourages inquiry into the possibility that our wax worm larvae, Medfly larvae, naval orange worm larvae, or black soldier fly larvae are crowded enough that 1) the O2 in their rearing container is chronically and substantially under the 21% (closer to 20.85%) of outside air? 2) the response of the respiratory structures is an increase in tracheole structures and mitochondria is to increase significantly over that found in “normal atmospheres”)? 3) if there are changes in the tracheoles and mitochondria, does this affect the fitness or quality of the insects that we are rearing? and 4) does the change in fitness or quality adversely affect the purpose of our rearing these insects? Other accessory questions could be 1) does the diversion of biomass to tracheoles and mitochondria come at the expense of other structures or fitness characteristics (wing size, cuticle resistance to microbial penetration, muscle activity potential)? 2) If the oxygen (and CO2) levels are abnormal due to the crowding and/or the gas exchange between the rearing containers and the rearing room air, is it possible that other density-related factors are askew (excess nitrogenous waste, enhanced microbial growth, deteriorated diet components)?
Of course, no rearing facility has the capacity to do all the inquiries that would be needed to answer all these (and MANY MORE) questions, but doing some critical thinking and data based inquiry can be derived from this type of thinking and respecting the complexity of the insects in our rearing systems!
In a recent post, I discussed the components of rearing systems that comprise the ecological niche setting. In that post, I provided a table from my book on rearing systems, and this table included 22 parameters which are present in the insects’ natural ecological niche and which must be provided in the artificial niche. For example, the table includes what I have called the “starter insects’ genetic characteristics” as well as food, waste product accommodations, gas exchange, moisture exchange, light relations, and so on.
In my experience with scores of rearing systems, small and large scale, many of these “niche components” are often neglected or underestimated by rearing personnel. Because most of us who require insects for research or other purposes are concerned only with the practical matter of having the insects available for our purposes, we do not bother to scrutinise the various features of how well our rearing system satisfies the fulfilment of the niche requirements. This is to say, we do not examine the character of the light relations (optimal photo-period, optimal intensity, and optimal light qualities or wavelengths). Likewise, we do not pay much (or any) attention to the microbial profile of our insects; and with few exceptions, we take at face value the microbial relations that are taking place in our insects. If we produce successive generations of Drosophila, for example, to meet our genetics investigations, we do not ask about the microbial profile of the insects’ guts or their environmental microbial associations. If the flies reproduce in their rearing containers, we do not ask if the gas exchange is optimal or if there is a stress imposed on our flies by excessive buildup of CO2 or by O2 deficits. Despite the fact that some researchers such as the VandenBrooks team have shown that O2 deficits can lead to dramatic changes in the respiratory system’s ultrastructure (increased tracheal branching and diameters as well as increased mitochondria associated with hypoxic conditions).
We rearing specialists and users of reared insects too often make the mistake of taking at face value the health and well-being (FITNESS) of our insects. Another way of regarding this fitness is as the QUALITY of our insects, and this is directly associated with the physiological concept of HOMEOSTASIS.
The above image (Figure 1) shows pre-pupal larvae being reared on a wheat germ diet derived from the Yamamoto 1969 tobacco hornworm diet. In this figure, a few of the “moving parts” of the rearing system are depicted, the insect, the diet, frass, and slits in lidding for gas exchange. There is also an indication that microbes are ever-present in rearing systems, within the insects, in the frass, and other parts of the rearing system. Still more features are present in this rearing sub-system, including the environmental conditions such as temperature, humidity, light qualities (including photo-phases, light intensity, and light quality). Sounds and odors also help comprise the insects’ environment as are container configuration and population density (number of individuals per unit of container volume). Many examples of the potential complexity and intricacy are prominent features of rearing systems that are helpful to understand; take microbes, for example.
Various microbial interactions are possible in the rearing system.
In Figure 2, the greenish background is a colony of fungi that grew on an insect diet where the moisture content was too high. In the upper foreground, the black and white image shows the partially-dissected gut of a lacewing adult with the gut contents shown to include yeast which are part of the rearing diet of the lacewings and which also reside in the insect’s specialised, diverticulum-based gut where the yeast thrive as symbionts with the host adult. The lower left picture shows whitefly larvae with their characteristic mycetomes (paired reddish structures), and the image on the lower right shows microbial inhabitants of the gut of termites. Besides these relationships of insects with symbionts (which are mutualists with the insects) and contaminants, there are also possible relationships with pathogenic microbes. ALL of these relationships must be understood to help keep our insects thriving (unstressed and displaying homeostasis) in the colonies that we house in our rearing systems. More on this in the next post. Please note the table (taken from Cohen 2021: Design, Operation, and Control of Insect Rearing Systems, CRC Press). I will comment further on this table in the next post.
Figure 6.1. Simplified Niche Parameters Expressed in an N-Dimensional Hypervolume. Figure 6.1 depicts the idea that there are many facets of niches, represented as intersecting or overlapping vectors. The intersections represent the concept that niche parameters are interactive.
Range of Niche Parameters
Starter Insects’ Genetic Characteristics
Full genome characteristics, including genetic diversity
Type & amount of food eaten
Nitrogenous wastes, moisture, undigested food
CO2 output, O2 intake
Water ingested, evaporatedInsects’ effects on humidity in rearing container through cuticular, respiratory, and fecal water loss, cuticular and respiratory water loss, osmotic values of water in aquatic systems
Effects of photoperiod, light intensity, and light quality (including possible other electromagnetic spectrum wavelengths such as UV and IR
Beneficial microbes, pathogens, commensals, etc.
How many individuals/unit of rearing space?
Gregarious vs. solitary, agonistic behavior, competition, group digestion, group thermal regulation, etc.
Effects on substrate (biting, lidding, etc.)
Degrade container by biting holes, webbing to “manage” frass, digging and conditioning soil, pH of water for aquatics, etc.
Ratios of ♂/ ♀; number of adults, environmental conditions, space for courtship, pheromone plumes, etc.
Materials for egg inserters, conduce conditions for egg-laying & egg development/egg hatch
Populations of competitors (e.g. mites and psocids)
Often on plants used to rear insects and sometimes on artificial diets (see text discussion for details)
Populations of parasites/predators
The large biomass and populations of mass-reared insects offer a target for cryptic parasitoids and predators
Temperature tolerances and optima influence the success or reared insects and further influence susceptibility to microbial attacks
Multifunctional uses of silk are discussed in the text
Effects of other (than silk) secretions: extra-oral fluids
e.g., glandular secretions, oral/salivary secretions, modifying nutritional quality and bioavailability
Scales (as dust hazards, etc.)
Potential for microbial transfers, influencing gas exchange, debris
Nitrogenous wastes as toxins, substrates for microbial growth, blocking normal food ingestion by clutter, etc.
Range of O2 uptake and CO2output on cultured insects, dispersal of O2 and CO2 in containers
Factors that interfere with auditory communication (stray noise) or vibrations that produce stress or induce excessive responses
Factors involved in oxidative stress
Factors in diet or environment that generate free radicals or reactive oxidative species in insects
“Design of experiments (DOE) is a systematic, efficient method that enables scientists and engineers to study the relationship between multiple input variables (aka factors) and key output variables (aka responses). It is a structured approach for collecting data and making discoveries.
“When to use DOE?
To determine whether a factor, or a collection of factors, has an effect on the response.
To determine whether factors interact in their effect on the response.
To model the behavior of the response as a function of the factors.
To Optimize the response.
“Ronald Fisher first introduced four enduring principles of DOE in 1926: the factorial principle, randomisation, replication and blocking. Generating and analysing these designs relied primarily on hand calculation in the past; until recently practitioners started using computer-generated designs for a more effective and efficient DOE.”
The quotation from the stated website (from JMP) gives the basis of the arguments that I am asserting about why DOE is such a valuable tool for insect rearing personnel. First, the idea of studying the relationships between multiple factors should appeal to every rearing specialist. When we put our insects into a rearing system and provide them with artificial diets, we should wonder how the diet components affect the insect and how they affect one-another. For example, how does the pH of the diet relate to solubility of a protein supplement such as casein? Furthermore, how does the pH affect solubility of salt mixtures such as Wesson salts? A little inquiry about casein (which should more properly be called “caseins” because there are several milk proteins that are indicated by the term “casein”) shows that this protein is very hard to dissolve at near neutral pH levels. Therefore, unless we make special efforts to dissolve casein (such as pre-dissolution in basic solutions), this important protein is likely to form pockets within the diet where it is non-homogeneously distributed. The casein-rich vs. casein-poor pockets contribute to the problem of non-uniformity, which leads to serious differences in growth and development rates of larvae that are otherwise similar in age and genetic composition. The same type of non-homogeneity applies to dissolution and distribution of salt mixtures. I have shown (Cohen 2015) that Wesson salts are virtually non-soluble unless they are dissolved in a low pH solution. That helps explain some of the positive effects of using diets that are on the acidic side, rather than neutral. Further analysis helps us understand that the texture of the diet, in terms of firmness or gel strength, is enhanced by the presence of salt mixtures where Wesson and Beck salts have an enhancing effect on the gel strength of agar, carrageenan, or other hydrocolloid gels (Cohen unpublished data).
In Figure 1, I have tried to suggest some of the complicated interactions that are present in an insect diet. The lesson here is that using DOE-based investigations, we can begin to dissect the components of our rearing systems to help us understand the ways that rearing factors affect the nature and success of our rearing systems. The whole thrust of this approach is to turn the “black boxes” that describe our rearing systems into more transparent and logical processes.
Currently, in all my classes, I use design of experiments (DOE) to help us better understand the factors that are most important in insect rearing systems. I originally became aware of the applications of DOE through a criticism of my statements about using one factor at a time for diet development in my book on insect diets (Cohen 2004). The criticism came from Dr. Steve Lapointe et al. (Lapointe, S. L., T. J. Evens, and R. P. Niedz. 2008. Insect diets as mixtures: Optimization for a polyphagous weevil. Journal of Insect Physiology. 54: 1157-1167.) I was initially dismayed at the criticism of my conventional (and outdated) thinking that we could only use one variable (one factor) at a time in a good scientific experiment. Once I got over the ego-deflation of having a blatantly narrow statement in my book, I set-out to learn how multiple factor experiments could be far more telling about the nature of our diets and other rearing system components.
As a start in my new-found application of DOE, I included this more sophisticated approach in the 2nd edition of my Insect Diets: Science and Technology, but I still had a steep learning curve. After years of effort to learn how to use the JMP approaches to DOE, I have reached a point where I routinely use various DOE tools (response surface designs, full-factorial designs, mixture designs, and Taguchi designs–all of which have their own specific applications). I have included extensive tutorials on using JMP’s system of DOE-based programs in my newest book, Design, Operation, and Control of Insect Rearing Systems (2021, CRC Press).
I want to make a special point about using JMP’s DOE: I am a physiologist, NOT A STATISTICIAN. Despite my trying to learn how to apply DOE, once I learned how powerful it could be in helping me understand the science behind insect rearing system dynamics, it was still a steep learning curve for me. I realize that most of my fellow rearing professionals are NOT statisticians either, and that is why I believe the JMP system’s DOE is so helpful. It allows people like me to ease their way into the very powerful statistical programs that can be so helpful in understanding what is going on in our rearing systems. The following mini-case study helps illustrate this point:
In Figure 1, I present a test that I did with painted lady butterflies (Vanessa cardui). In this test, I was asking the questions about the efficacy of sweet potato flour, rice bran extract, citric acid, and three types of protein (casein, soy, and dairy whey). The table in Figure 1 shows that I used two levels of sweet potato flour, two of rice bran extract, and two of citric acid while I provided casein, soy, or whey proteins. This full-factorial design required that I make up and evaluate 24 different diets (all of which had the same proportions of the other diet components not shown here). In the last two columns (on the right), the responses of total biomass per rearing unit and mean weight of individuals are presented. Feeding these data into the JMP system resulted in a complete statistical analysis of the experiment, including this convenient summary of the outcomes:
In Figure 2, the summary of the experiment is shown in terms of the total biomass that was derived from insects on the 24 different diets. This graphic representation helps us see clearly that the higher amount of sweet potato flour (10 g) gave superior results to the 5 g versions. The rice bran extract did not contribute substantially in either the 3 g or the 6 g versions. The higher level of citric acid (2 g) gave FAR better results than the lower (1 g). And the three proteins were not very different from one-another, with a possible trend towards the whey protein. What is not shown here (for reasons of simplicity) is that another graphic table in the JMP results shows which factors had interactions with other factors (positive or negative interactions). And this is one of the most exciting parts of using design of experiments with multi-factor components. It is, in my estimation, the interactions that really help explain how our rearing systems work.
By using a DOE approach we can better explore what factors are of significance in our rearing systems, the direction of their influence (positive or negative), and the factors which interact in ways that would not be intuitively predictable. Therefore, my teaching approach is to examine our rearing systems through the DOE approach! More about this will be presented in subsequent blogs and web pages.