We Rear Insects

Why Use Design of Experiments (DoE or DOE)?

Instead of studying one factor at a time, rearing specialists can inquire about how multiple factors (or experimental variables) function within an insect rearing system. By doing DOE procedures, we can save on costly runs for our experiments; AND we can discover interactions between factors that we could not understand by using one-factor-at-a-time (OFAT) experiments.

Some History of DOE in Engineering and Science and Specifically in Insect Rearing Science:

After struggling for almost 25 years (1979-2003) to develop artificial diets for various insects, I wrote the book Insect Diets: Science and Technology in 2003 (CRC Press, Boca Raton, FL) where I had stated that insect diet experts were burdened with experimenting with one factor at a time in their need to conform to “proper” experiemental design, which demanded a single factor or variable that was responsible for an outcome/effect/response.

Soon after my pronouncement about one factor at a time experiments, Dr. Steve Lapointe and his associates published a paper that was revolutionary for me and in my opinion for the entire community of insect rearing specialists: (Lapointe et al. 2008) where Lapointe and his team stated the reality that insect diets were mixtures and as mixtures could be treated with the statistical frameworks that described and helped analysis of complex mixtures and combinations of other interacting factors. Frankly, when I read Lapointe’s bold assertion that things could be done much more parsimoniously and effectively with multiple factor experiments, I was hurt and embarassed that my seemingly authoratative pronouncement was incorrect and naïve (though the authors were gentle with my assertion, I still felt a sting from having my authority challenged). After “licking the wounds of my pride,” I started to ask what was Lapointe talking about—doing experiments with multiple factors—using several variables in one set of experiments?

Reference: Lapointe, S. L., Evens, T. J. & Niedz, R. P. Insect diets as mixtures: Optimization for a polyphagous weevil. J. Ins. Physiol. 54,1157–1167 (2008).

In this historically important paper, Lapointe et al. used a response surface design to test multiple factors to improve a diet for Diaprepes a weevil that had proved difficult in prior efforts at diet development/improvement. The group showed that using the response surface design greatly simplified diet development, but beyond the importance of the accomplishment for a single insect the Lapointe group showed that DOE was a most valuable tool in diet development and for that matter rearing system development and improvement in general.

This set me into motion to try to learn how DOE worked and how I could apply it to my rearing/diet development goals. I will tell that story and my walk through the DOE system that I have been using: the JMP statistical package. Please see the following blog pages in the next few days….

Reminder: Professor Allen Carson Cohen will be teaching a live, virtual course, using Zoom, starting June 3, 2025 through July 10, 2025. Classes will go from 10:00 am (1000 hours) through 12:00 noon (1200 hours) each Tuesday and Thursday. Registration cost will be $450, and classes are taught as continuing education courses through the North Carolina State University’ Office of Continuing Education and Professional Development.

To register for the class or to get more information, please use this link: https://lifelonglearning.ncsu.edu/reporter_course/insect-rearing-system-fundamentals/

Also, for further information about registering or other course matters, please contact Mr. Jamie Merritt, Program Coordinator at this email address: [email protected] or Ms. Darthea Powden at this email address: [email protected]

For any further information about the course contents and issues regarding your expectations, please feel free to contact Professor Allen Carson Cohen: [email protected]

This diagram represents several features to be covered in the upcoming course.

Classes are taught as live lectures and discussions with participants encouraged to interact with Professor Cohen. The live presentations allow interactions between participants and with materials that include PowerPoints, videos, demonstrations, and many resource materials presented through the highly-touted Moodle system.

Please check the next blog page for May 15-16 on using design of experiments as a basis for developing and improving rearing systems.

To view the video in this tutorial example, please right click the image and select “Show Controls,” then click the forward arrow to view the video.

The special feature of this approach is that collaborators or teacher/learners can communicate live through Zoom or other remote formats and share ideas, explanations, etc.

This is an example of a tutorial where the collaborators (Professor Cohen and Dr. Carole Cheah) were exchanging information about diet development for their project dedicated to development of artificial diets for predators that are specialists of hemlock woolly adelgids. In this video, recorded from a video collaboration, Drs. Cheah and Cohen review various aspects of the acceptance of the artificial prey (artificial diet later published in a 2015 paper (published here: Cohen and Cheah, Entomol Ornithol Herpetol 2015, 4:2 http://dx.doi.org/10.4172/2161-098)

The main point of this blog page is to illustrate the potential quality and efficiency of remote communication (Cheah was in Connecticut and Cohen was in North Carolina for this Zoom meeting). Note that the video here includes only Dr. Cheah’s part of the communication where Cheah showed Cohen videos and still shots of the beetles she was feeding diets produced by Cohen as freeze dried samples of their “CC Diet.” Dr. Cheah was testing the responses of the beetles to the diets with various additives and diet presentation conditions such as on or off of hemlock foliage or Parafilm or other substrates. Please note further that Dr. Cheah at the time was using a simple phone-camera and shot the videos and stills through a dissecting scope eyepiece.

This type of exchange works well for many other kinds of information exchanges which can be done live and which can further be made to include multiple research or rearing education partners.

Cohen is currently offering this type of collaboration and teaching as part of an online experience for collaborators or participants worldwide.

MORE TO FOLLOW SOON!

This post and several others to follow are intended to narrate how I approach teaching about insect rearing systems and how I approach developing and improving these systems. I am writing this page on February 14, 2025 almost 50 years after I started out in insect rearing at the University of Arizona and the USDA, Agricultural Research Service in August of 1979. During this time, I have had several successes and several failures at developing and/or improving rearing systems. It has become the focus of my life to better understand why systems and the components of those systems work or fail. These pages are my effort to explain what I have learned in trying to understand the scientific principles behind rearing.

First, the concept of the rearing system as an ecological niche: before I was an entomologist/rearing specialist, I was an ecologist. Over the years, I have come to appreciate the reality that as rearing specialists, we are obligated to provide all the components that our insects would have in nature–only we must put them all into our rearing system. THIS IS TO SAY THAT OUR REARING SYSTEMS ARE ARTIFICIAL ECOLOGICAL NICHES!

As “NICHE-KEEPERS,” we must provide the food, the gas exchange, the heat, light, humidity, sites for oviposition, microbial relations, mating sites, oviposition sites, etc., etc. Are we giving our insects the right conditions to safely void their urine and fecal wastes? You watch a leaf-eating caterpillar in nature dropping its frass from the leaf-feeding site to the ground. How must they release their wastes in our containers? We know what is convenient for us as the rearing personnel, but is that what is best for the insects?

The two pictures in this post show a little of what I mean by factors in the ecological niche concept of insect rearing systems. The top picture is an attempt for me to show in multiple dimensions (based on G. Evelyn Hutchinson’s “N-Dimensional Hypervolume” as the model of an ecological niche). The idea here is that CO2 generation and O2 uptake are not only related to one-another, but they relate to the thermal conditions and the food the insect is eating. The ratio of CO2/O2 or the respiratory quotient are indicators of whether the insect is metabolising mainly carbohydrates, lipids, or proteins. If being a little confusing with all this and other factors seen in the top diagram, it’s what I intend to do to convince you that this is all pretty complex and VERY INTER-RELATED stuff.

The second picture showing the diet (developed by R.T. Yamamoto 1969) for Manduca sexta is also a complex of components such as wheat germ, casein protein, torula yeast, vitamins, and minerals that are all interacting with one-another to give the diet a composition, texture, consistency, and configuration that help determine the success of our tobacco hornworm rearing system. One reason that the diet is (in my opinion) so successful for M. sexta is that it provides lipids (fatty acids and sterols, for example) in the form of lipoproteins which make these essential lipids bioavailable for the larvae. One of the wonderful features of wheat germ is that it contains nutritious proteins with all the essential amino acids, but also some of these proteins are lipoproteins, which carry the lipids in a bioavailable way compatible with our hornworms’ digestive system.

More along this line in my next blog entry. Happy Valentine’s Day!

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!