1. Introduction
Wheat flour is one of the most common types of flour processed and consumed around the world but there is a growing market for alternatives to wheat flour [
1,
2]. The market for alternatives to wheat flour is growing substantially as coeliac disease diagnoses increase and consumers demand low gluten and gluten-free options [
1,
2,
3]. In 2007, the gluten-free market in the United Kingdom was valued at GBP 74 million, roughly USD 93 million and in 2006 the market in the United States was valued at USD 696 million (1). In 2019, the gluten-free market was valued at USD 21.61 billion and is expected to grow by 9.2% by 2027 [
4]. Alternatives can include other grains such as sorghum or corn but also include an expanding number of flours made from other foods, including coffee, banana, nuts, and root vegetables such as cassava or potato [
3]. After the grain is harvested, it is stored and processed and vulnerable to insect pests that can damage or destroy the commodity and can cost producers billions of dollars in lost revenue each year. Traditional cereal grains such as wheat and rice have been studied extensively as to how insects can grow and develop on whole seeds, milled flours, and milling fractions and byproducts [
5,
6]. However, as the diversity of flour types used by the food industry continues to grow, less is known about the ability of stored product insects to develop on these alternative flours and the potential risks these insects might cause to this rapidly growing industry.
Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) is a major pest of wheat and rice flour [
7,
8] and a secondary pest feeder of stored products and has been documented to survive on a variety of commodities. These insects have high fecundity on wheat flour and survive well on a variety of wheat strains [
9]. A diet rich in folic acid aided in growth and reproduction for
T. castaneum [
10] and low levels of phosphorus (0.075%) in the diet were insufficient for population growth while high levels (3.075%) were toxic to the related
T. confusum Jacquelin du Val (Coleoptera: Tenebrionidae) [
11]. Yeast added to wheat flour increased reproduction and survival of
T. castaneum [
10] and beetles had better fecundity than on plain wheat flour, millet, barley, rice, sorghum, and soybean, with sorghum and soybean flour having the lowest fecundity [
12].
T. castaneum is an important pest on wheat flour, but it has a broad host range and has been reported to be associated with diverse commodities such as rice, millet, sorghum, and corn [
13,
14]. For many of these commodities, the information available on development and fecundity is limited.
T. castaneum had high progeny output on millet, sorghum and maize meals [
13], high progeny output and low mortality on starch-rich pea flour [
15], atta flour, wheat flour, self-rising wheat flour, rice flour [
16], whole wheat flour, corn flour, and brown rice flour [
17]. In comparison,
T. castaneum had poor progeny production on semovita (a brand of semolina or durum wheat), cassava or yam meals [
13], protein-rich pea flour [
15], soy flour [
17], tapioca, and potato starch [
16], and poor feeding and development on cowpea flour [
18]. In addition, comparisons of wheat or barley varieties and milling fractions showed variable results for
T. castaneum. There was a range of fecundity and hatch rates on different varieties of barley [
19] and developmental times increased on pelletized frog feed and crumbled poultry feed [
20] and larval weight gain was slow on dried distiller’s grain [
21]. Rice flour milling fractions had varying progeny output with higher progeny output on rice flour, milled whole rice kernels, brown rice, milled broken kernels and rice bran but lower progeny output on rough rice hulls, paddy rice dust, and milled rice dust [
5] suggesting that not only does the commodity matter but also the size and shape of the commodity. In addition, commercial flour products of corn, rice, wheat, and soybean had lower population density but quicker developmental rate than non-commercial flour of the same commodity [
22]. These studies indicate that flour type makes a difference in insect reproduction and growth, but few studies have assessed a wide range of flour types under the same experimental conditions.
Understanding how T. castaneum oviposit on different types of flours and how progeny develop over time will provide information on the suitability of different alternative flours. Here we evaluated almond, amaranth, barley, buckwheat, cassava, coconut, corn, garbanzo, millet, oat, potato, quinoa, rice, rye, sorghum, spelt, and teff flours compared to wheat flour (as a control). Where possible we obtained these flours from the same two commercial sources to determine how source impacted suitability. Here we examine how many eggs T. castaneum females lay on the different flours and how successfully these progeny develop to the adult stage. We also assessed development under a more standardized set of conditions by placing single eggs on each flour and measuring the ability to develop to the adult stage. These two tests enable a look at the female’s recognition of each flour as a suitable oviposition substrate and the quality of the resource for progeny development.
4. Discussion
The type of flour had a significant effect on the number of eggs laid and progeny produced. The differences in oviposition are likely driven by volatile olfactory cues, textural cues, or chemical gustatory cues. Flours that had a low risk of infestation had a distinctly different texture than other flours tested which was more powdered and finer or were oily with a semi-flaky texture. In addition, nutritional content such as sodium, fiber, or protein can drive infestation risk. For example, flours with high caloric content had no progeny emergence. Examples of low risk of infestation flours from our study include cassava, amaranth and potato which had a more powdered and finer texture and coconut and almond which were oily with semi-flaky texture compared to the other flours tested. In addition, coconut and almond had high caloric content. In
Manduca sexta high-fat diets also led to higher mortality and lower body weights [
23].
Chemical and physical properties of these flours have been shown to have a significant impact on the development of insects. For example, both corn and wheat flour supplemented with brewer’s yeast did significantly better in productivity than just corn and wheat flour without yeast [
17]. Similar to our results showing a positive association with egg count and protein, Astuti et al. [
22] showed that in the flours they tested, flours with low protein content (<5%) were unsuitable for
T. castaneum development but also that high protein content in flours like soybean flour (>25%) was also unsuitable for development. Wong and Lee [
16] also detailed a trade-off of carbohydrates and proteins within flours for growth and fecundity. Our results suggest that protein also plays a role in successful progeny emergence and oviposition, with increasing risk of infestation with increasing protein content, but the range of protein content tested (0 to 15.5 g) is not a function of the percentage of the diet, and is not a large enough range to make a significant association of a maximum protein level.
Particle size, which we hypothesize to play a role in our series of flours tested, has been shown to play a significant role and can impact the microclimate of the
T. castaneum [
20,
21,
22] but was not explicitly tested here. However, flours at high risk for infestation are all types of traditional grains with similar nutritional properties and textures. For progeny emergence, rye, wheat, millet, and teff flours had the highest numbers of progeny emerge while buckwheat had moderately high progeny emergence. Sorghum, corn, and rice had relatively low numbers of progeny emerge and suggests something likely nutritional or chemical within the flour is affecting development to adult stage post-egg laying. However, sorghum and corn had over 75% of single eggs develop into adults 8 weeks after eggs were placed on the given flour. A possible explanation for this difference between progeny laid emergence and single egg development could be that additional nutritional properties that were sieved out for egg counting purposes are needed to fulfill the specific nutritional components for egg development in
T. castaneum.The interaction of flour type and source also had a significant impact, but sources were typically equal in progeny and egg counts, and differences were only detected for the number of eggs laid for corn and in adult progeny counts for oat and millet. The overall similarities between sources suggest that regardless of the source of the flour, the infestation potential of these insects can be considered consistent based on the flour type. The differences in egg counts for corn could be due to the two corn flours being different varieties as Source 2 was snow-white corn and Source 1 was yellow corn. In addition, the differences between progeny counts for millet and oat could be due to Source 2 millet and oat not having eggs counted, thus reducing any damage to eggs laid and increasing progeny emergence. However, eggs laid in laboratory wheat flour had high progeny emergence of over 80% and single egg development to adults was about 75% for wheat flour, so experimental damage is unlikely to reduce progeny emergence significantly. Differences between sources could also be due to source or plant variety differences in nutrition, source of grain as in where it was grown and how it was processed, or potential contaminates.
Flour type in addition to impacting the probability of development to the adult stage, also impacted the time it took to develop. In addition to sorghum and corn having high single egg development to adult, teff, wheat, barley, garbanzo, millet, rye, spelt, and buckwheat also had over 75% of single eggs develop into adults 8 weeks after eggs were placed on a given flour, although the timing of adult emergence was different among these flours. For example, by week 6 after eggs were placed on the flour, teff and wheat both had at least 70% of eggs developed into adults while it took garbanzo 8 weeks to get 75% of eggs to adults and barley and buckwheat 7 weeks to develop 75% to adults. These results suggest that although these flours have high adult emergence success and must contain all nutritional requirements, some flours, such as garbanzo, may have the necessary nutritional components in lower amounts that take a longer time to accumulate to trigger adult emergence. For example, riboflavin has been implicated in the development of
T. castaneum [
17,
22,
24] and although not measured or available on package labels here, low riboflavin levels could be a factor in the increased developmental times or lack of adult emergence.
Further, nutritive content likely played a significant role in the overall number of eggs laid and overall progeny emergence. For example, the higher the sodium content, the fewer eggs were likely to be laid in each flour while higher fiber and protein resulted in a greater number of eggs laid. For progeny, higher carbohydrate content resulted in more progeny that emerged, while higher calories from fat, saturated fat, and sodium resulted in lower numbers of eggs laid. Our analyses show that higher sodium tends to lead to lower numbers of eggs and progeny emergence. This could be due to higher sodium levels acting as a deterrent to laying eggs as shown in blowflies [
25] or too much sodium resulting in decreased larval development [
26]. Higher fiber and protein levels also tended to result in increases in eggs laid and progeny emergence but high protein levels can also act as a deterrent in some species [
27] or inhibit progeny emergence [
15]. Overall, a more detailed analysis of components of these flours will allow a finer scale analysis of specific nutritive components crucial for
T. castaneum growth; interestingly, differences between sources also create further questions as to what is differing in flours from different sources.
Similar to single egg success showing differences in hatching and adult emergence timing, estimates of curves for progeny emergence over time showed clustering of flours with different timing patterns. Again, teff, millet, wheat, spelt, and barley showed patterns consistent with their high numbers of eggs laid and progeny emergence and had progeny emerge early in high numbers. There was a significant splitting of sources again for flours such as garbanzo, oat, and sorghum. For example, garbanzo and sorghum from Source 2 had slower or delayed emergence of progeny compared to garbanzo and sorghum from Source 1. If we remove sources with slower emergence rates, we can model curves that predict the quickest that the flour will be at risk of adult emergence. Teff, millet, wheat, barley, and spelt are at risk earliest for adult emergence, while corn, buckwheat, garbanzo, oat, rye, quinoa, and rice had early progeny emergence but tended to have lower numbers of adults. Gauging infestation potential or risk based on these flours can provide a good estimate of when to potentially stop further generations from infesting the product. Flours with late progeny output such as potato and sorghum would not provide a good predictive estimate of infestation until it may be too late to stop a major infestation, especially if there are other flours stored in the same area.
In addition to the developmental timing, the overall success of egg hatching and timing of this hatching is also impacted by flour type. Estimating when 50% or 90% of eggs hatch or adults emerge is important to predicting the overall success and potential of mating populations within a product. Almond, amaranth, and coconut are predicted to take the longest to hatch to larvae but our models predict years for these beetles to emerge as adults, in which time they would most likely die as larvae. Similarly, amaranth, cassava, and potato are predicted to take years to emerge into adults. Eggs on most flours are predicted to take less than 4 weeks for both 50% or 90% of them to hatch. Sorghum, spelt, teff and wheat were quickest for 50% of adults to emerge and sorghum, buckwheat, corn, spelt, and barley were the quickest for 90% of adults to emerge. These flours with the quickest emergence time could be used as indicators of infestation in a warehouse or storefront setting.
Unconventional flours made from a variety of crops have been receiving attention from a consumer standpoint [
1,
2,
3,
4] but the risk of infestation of stored product pests has received limited attention. Main diet staples specific to a given region of the world such as yam, cassava, semovita [
13], corn, rice, mung bean, sago, and breadfruit [
22] have been studied to determine how insects can grow and infest on these important local commodities. Similarly, major pest species in different areas such as
Prostephanus truncatus or
Trogoderma granarium have also received specific attention due to their detrimental effects in a given region in the world [
28,
29]. However, a study with a broad examination of flours like the one detailed in this manuscript can provide a baseline for establishing infestation risk thresholds and monitoring of a cosmopolitan pest on a range of different products. As options for gluten-free or enhanced nutrition flours grow [
1,
3], risk assessment for insect pests is necessary for developing integrated pest management programs and solutions to protect these high dollar commodity products. This research suggests that there are several factors that may result in a lowered risk of insect infestation such as sodium content and particle size and further research needs to be completed to determine what other nutritional factors and physical characteristics can deter both oviposition and development of insect pests.
In our study, we examined the relative risk of
T. castaneum on a variety of different flours, but other stored product pest species may have different patterns in terms of the risk they pose to these flours and patterns of competition among species may also differ on different flour types [
30,
31]. Other species could have vastly different responses to these flours tested compared to
T. castaneum and strain-specific dietary preferences and toxicity effects for a single species could significantly impact infestation risks [
32,
33]. In addition, understanding how communities of insects interact with local food sources is critical as the abundance and variety of these commodities are changing and shifts in community composition could drive increased risk in product infestation as other minor pests become more problematic.
Our study did not provide the beetles with a choice of flour source to oviposit eggs, but given a choice, females may respond differently to volatile, chemical, or textural cues associated with each flour. These choice studies must be examined in the future to determine how insects survey their environment and make decisions on oviposition and development sites for their offspring. Understanding how insects choose their oviposition sites will also provide management information for mixed storage sites, where, for example, wheat flour and cassava flour are stored near one another. In these scenarios, one might assume that if the wheat flour is infested with insects, the cassava flour must be infested too and therefore, all these flours must be treated for the insect. However, it is likely that the cassava flour will not be infested and does not need to be treated. In addition, it may be valuable to store high dollar flours near high risk of infestation flours and insects may choose to infest the “bait” flour and not the more valuable flour. Future choice experiments will provide further information on the decisions for oviposition in these insects and will provide further information on how to behaviorally manage these pests. In addition, understanding which components of these flours can act to either inhibit or promote oviposition and the development of insects in these flours can be used as a reduced risk tactic.