In a recent post (November 30, 2016), I discussed some of the foundations of Drosophila rearing, and I pointed out that much of the rearing that was done over the past century depended on the early works with Drosophila. In fact, the modern concepts of insect rearing must have originated in the 1910 paper by Delcourt and Guyenot: “The Possibility of Studying Certain Diptera in a Defined Environment.” This title, to me, represents and suggests the concept of CONTROLLED REARING.
Baumberger, J. P. 1917a. The food of Drosophila melanogaster Meigen. Proceedings of the National Academy of Sciences of the United States of America: 3: 122-126.
Baumberger, J.P. 1917b. Solid media for rearing Drosophila. American Naturalist. 51: 447-448.
Delcourt, A. and E. Guyenot. 1910. The possibility of studying certain Diptera in a defined environment. Comptes rendus hebdomadaires des séances de l’Académie des sciences (0001-4036), 151, p. 255-257.
Guyenot, E. 1913a. A biological study of a Drosophila ampelophila Low fly I – The possibility of an aseptic life for an individual and the line. Comptes rendus des séances de la Société de biologie et de ses filiales (0037-9026), 74, p. 97-99.
This is a “mini-review” of a new paper that was published on “Genetic and microbiome changes during laboratory adaptation in the key pest Drosophila suzukii. The authors of this paper (K. Nikolouli, H. Colinet, C. Stauffer, and K. Bourtzis) have made this important contribution in the journal Entomologia Generalis, Volume 42 (2022), Issue 5: pp 723-732, and they track wild D. suzukii from the field through multiple generations of laboratory culture. Nikolouli et al. point out that the assumption that field-derived insects make profound changes in their various adaptations, including genetic diversity and symbiotic community changes as they adapt to the artificial conditions of rearing. The authors summarize their work reported in this paper with the following statements “These results can serve as a reference for the design of an area-wide integrated pest management approach with a Sterile Insect Technique (SIT) component. Rearing productivity, biological quality, and mating competitiveness of a SIT mass-reared strain should be assessed in connection with genetic and symbiotic changes occurring during laboratory adaptation.“
The authors statements about the SIT context of these findings actually goes far beyond sterile insect technique, and the findings about changes in genetic characters of the target insects AND the microbiomes have profound implications for rearing for all other purposes, including biological control, insects as food and feed, and research.
A central tenet of the rearing program at NCSU is that insect rearing systems are artificial ecological niches of the insects we rear. This means that every aspect of the insect’s needs must be met by our rearing system. This responsibility of rearing personnel applies whether we know each requirement or not. For example, each insect (and each insect population in our colonies) requires a certain range of oxygen to meet their metabolic needs. We may not know how much oxygen this requires (the range of oxygen concentrations), but when we provide holes in the lid of the rearing container or a screen that permits gas exchange, we hope that the openings are adequate to meet the oxygen demands.
Therefore, we can think of the oxygen requirements as part of our insects’ ecological niche, and if our insects perform adequately under the conditions of our rearing containers, we assume that everything is OK “oxygen-wise.” We make the same kinds of assumptions about carbon dioxide concentrations and air flow and the same about water vapour. Sometimes we get ourselves in trouble when we try to restrict water loss from he diet or the insects by making the openings so limited that we wind up starving our insects of adequate oxygen (a phenomenon known as hypoxia or even anoxia); and/or we make create a situation of excess carbon dioxide, which can threaten our insects’ well-being.
In all the rearing courses that I teach, I try to convey to students that the rearing system with all its components that meet the insect’s ecological niche parameters is a complex set of interactions between various (ALL) components, and a helpful way of viewing all this is with a 3-D model or diagram such as what is seen here:
In this diagram that simulates or suggests three-dimensional space, I have tried to show about 20 of the many, many parameters in a niche. The diagram is intended to illustrate that there are interconnections and interactions that involve all the biotic (biological) and abiotic (physical/chemical) factors that play roles in our insects’ well-being. This model of N-dimensional hyperspace is derived from the work of the famous ecologist G. Evelyn Hutchison. I have used this model in my recent book, Design, Operation, and Control of Insect Rearing Systems 2021, CRC Press, Boca Raton, FL).
For this discussion, let us take-up a simplification of this diagram with 3 factors, heat, CO2, and O2. Here is the simplified diagram:
In this 3-D, three-factor diagram, I am trying to show that the waxworm from my colony (of Galleria mellonella) is greatly influenced by the heat (temperature) conditions in their rearing container; but also, the concentration of CO2 and O2 are interacting factors that influence the metabolism and aggregation behavior of the waxworms, as well as food consumption, digestion rates, development rates, etc.
These interactions are illustrated in the images taken from my waxworm colony where I used a thermal imaging camera to capture the heat (thermal) gradient produced by the waxworms in this container.
In the study of the colony described here, I have also included (besides the thermal profile) the CO2 and O2 gradient that results from the waxworms’ behavior and metabolism. The example, taken from waxworm cultivation in my research program, is typical of the kinds of multi-dimensional dynamics that apply to ALL insect rearing systems. My whole point in this and related discussions is to get students of insect rearing to recognise the complexities of their rearing systems components. It is further intended to awaken students’ appreciation for knowing the nature of (the science of) these many factors and their interactions.
This approach is not simple or easy; but it is very powerful in establishing and maintaining healthy, productive colonies!
Based on feedback from our students from rearing courses over the past 2 years, we are modifying our approach to the online courses. We are now offering a new rearing fundamentals course for entry level rearing background and mid-level background. The course will cover all major aspects of insect rearing systems with emphasis on basics of how components of rearing system function and how they interact.
An important part of the course on Insect Rearing Fundamentals is to give students/participants a better understanding of how various insects feed and how this knowledge helps lead to better rearing techniques. In this example, typical fly feeding dynamics are shown with the larva (in the center) using its hook-type mouthparts to help ingest food after it has been treated by the larva with its digestive enzymes (extra-oral digestion). The adults, especially the one in the upper left, are using their lapping/sponging mouthparts to help them ingest the yeast-based diet and diet matrix of agar and plant materials. This type of feeding applies to many families and species of flies, including the tephritid fruit flies (such as Mexflies and Medflies), which are extensively treated in this course.
This is a sample of the kind of conceptual and detailed explanations that Professor Carson Cohen provides in his lectures. Here are the topics that will be covered in the 12 units (lecture/discussions) in this course:
April 4: Lecture 1: The rearing system concept
April 6: Lecture 2: Diets: Overview of insect nutrition and metabolism
April 11: Lecture 3: Diet components–chemical and physical interactions
April 13: Lecture 4: Diets: Diet-making and diet presentation
April 18: Lecture 5: Diets: Optimization of diet composition
April 20: Lecture 6: Diets: Matching diets with feeding biology
April 25: Lecture 7: Domestication/colonisation (insect genetics and epigenetics)
April 27: Lecture 8: Microbial relations in insect rearing systems
May 2: Lecture 9: More microbial relations (kinds of microbes and kinds of interactions such as mutualism and pathology)
May 4: Lecture 10: Reducing error and stress in rearing systems part 1
May 9: Lecture 11: Reducing error and stress in rearing systems part 2
May 11: Lecture 12: Process control and quality control of reared insects
Each unit will include a 90 minute lecture with a 30 minute discussion section. This structure will allow for generous time for questions and answers that pertain to students/participants’ specific learning needs.
Students/participants will be given access to the NCSU Moodle system which gives access to class notes for each lecture and readings that are supplements to the lectures. The course is not graded, but quizzes will help provide feedback for students/participants to know where they may need explanation from the instructor.
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 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