Temperature and relative humidity (RH)

Temperatures commonly used to rear mealworms are 25–28°C (Kim et al. 2015; Koo et al. 2013; Ludwig 1956; Punzo 1975; Punzo and Mutchmor 1980; Spencer and Spencer 2006,), with an absolute minimum of 10°C (Punzo and Mutchmor 1980) and a maximum of 35°C (Martin et al. 1976; Punzo and Mutchmor 1980). Temperatures <17°C inhibit embryonic development (Koo et al. 2013) and temperatures >30°C increase death rates (Koo et al. 2013; Ludwig 1956). The minimal and maximum lethal temperatures for exposure periods of 24 h are 7–8°C (Mutchmor and Richards 1961) and 40–44°C (Altman and Dittmer 1973; Martin et al. 1976), respectively. There are no significant differences in the temperature requirements for the different stages of development of this species (Table 2).

Table 2 Minimum, maximum, and optimal temperature (°C) conditions used on the rearing of eggs, larvae, pupae, and adults of mealworms.

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Mealworm response to RH varies, although their resilience depends largely on temperature (Table 3). In all studies focused on the influence of RH on the development of T. molitor, optimum values varied from 60% (Manojlovic 1987) to 75% RH (Punzo 1975; Punzo and Mutchmor 1980).

Table 3 Minimum, maximum, and optimal relative humidity (%) conditions on the rearing of eggs, larvae, pupae, and adults of mealworms.

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Temperature combined with RH affects both the number and the length of the instars and also influences the water absorption capacity of the different stages of the life cycle. The pupal stage is the most resilient to extreme conditions of temperature and RH while the egg and early larval stages are the most sensitive (Punzo and Mutchmor 1980).

Oviposition does not occur at temperatures below 14°C with an RH of 65%, but even at the optimal temperature of 27°C in low humidity (20%), oviposition is significantly reduced due to the loss of body moisture by evaporation and subsequent death (Dick 1937). Female adult activity is also reported to be greatest at 90–100% RH (Hardouin and Mahoux 2003).

At 10°C (Punzo 1975; Punzo and Mutchmor 1980) and 12.5°C (Kim et al. 2015), water absorption in eggs is reduced and embryological development halts. Whatever the temperature, extremely dry conditions of about 12% RH potentiate water losses from the eggs, culminating in death due to desiccation (Punzo 1975).

The growth rate of T. molitor larvae is highly dependent on moisture, with higher growth rates at 90–100% RH (Hardouin and Mahoux 2003) and even at 70% RH, but the rate slows at 30% RH and hardly occurs at 13% (Fraenkel 1950). During times of extremely dry conditions, mealworm larvae may cease feeding and become inactive until RH rises to more-favorable levels (Urs and Hopkins 1973). In general, the higher the RH, the higher the growth rate, but high levels of moisture favor the growth colony contaminants (e.g., fungi, other microorganisms, mites) (Fraenkel 1950).

Larval instar stages are shorter, and the numbers of instars are greater at 30°C than at 25°C, resulting in a longer total larval developmental period at 30° than at 25°C (Ludwig 1956). Temperatures below 10°C and above 35°C clearly constitute stress conditions for this species, although the minimal lethal temperature is 7°C and the maximum lethal temperature is 44°C (Punzo and Mutchmor 1980).

Tenebrio molitor development and survival are affected by the interaction of temperature and moisture. The effect of temperature is potentiated at extreme moisture conditions, and the effect of moisture is also more critical at extreme temperatures (Punzo and Mutchmor 1980). For example, at optimal temperatures of 25–27.5°C, T. molitor displays no stress even under extreme humidity conditions and long exposure periods. Moreover, while at 25°C the decrease in moisture has no significant effects on adults, larvae, or pupae, a temperature of 10°C increases mortality (Punzo and Mutchmor 1980).

Population density

Our search found that several densities of larvae have been used in experiments, depending on the objective of the study. These have ranged from 1 larva/dm² (i.e., a single larva in a 10-cm diameter Petri dish) to a maximum of 750 larva/dm² (Table 4). Morales-Ramos et al. (2012) studied the effect of density on adult population survival and production, obtaining an optimal density of 8.4 adults/dm² for mass production purposes.

Table 4 Physical conditions and population density used on the rearing of mealworms.

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Population density influences the number and the duration of the larval molts, with higher population densities resulting in fewer larval instars and smaller-sized penultimate larvae (Connat et al. 1991; Morales-Ramos et al. 2012; Morales-Ramos and Rojas 2015; Tschinkel and Willson 1971; Weaver and McFarlane 1990). Early studies have noted inhibition of pupation and cannibalism associated with crowding in tenebrionid beetles (Tschinkel and Willson 1971) as well as the occurrence of incomplete transformations and slower growth rates due to a reduced feeding opportunity induced by conspecific competition (Weaver and McFarlane 1990). Although high population density leads to a reduction in food consumption, the lower growth rates are also a consequence of the decrease in the efficiencies of both ingested and digested food conversion (Morales-Ramos and Rojas 2015). Overcrowding also reduces female progeny and reproductive output, with rearing of two insects per chamber being the most productive in terms of number of progeny produced per female (Morales-Ramos et al. 2012). Furthermore, overpopulation also affects the prepupal stage, with isolated larvae exhibiting accelerated larval–pupal apolyses and a reduced number of larval instars (Connat et al. 1991). Moreover, the metabolism of larvae in overcrowded populations results in a substantial increase in temperature that can easily be lethal.

Photoperiod

Mealworms are negatively phototropic and phototactic (Balfour and Carmichael 1928; Cloudsley-Thompson 1953), with adults and larger larvae positioning below the surface of the substrate during daylight and coming to surface in darkness. Although photoperiod influences growth and development of mealworms (Tyshchenko and Ba 1986), the response to photoperiod tends to disappear under constant conditions, when T. molitor becomes arrhythmic (Cloudsley-Thompson 1953). Recent studies show that larval development was optimal in long-day conditions, with lower development times observed in photoperiods of 14L:10D (Kim et al. 2015). Photoperiod also influences eclosion rates, with higher rates in long-day conditions, i.e., 45.5% at 14L:10D versus 24.2% at 10L:14D (Kim et al. 2015) and pupation with 100% pupation induced by a 12L:12D regime at 25°C. Under other photoperiod regimes, long-day result in more-protracted delays than did shorter-day conditions. However, at 30°C the photoperiodic response was reversed, with an inhibition of pupation under 12L:12D and enhancement under 18L:6D (Tyshchenko and Ba 1986) (Table 5).

Table 5. Photoperiod regimes used in the rearing of Tenebrio molitor.

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Oxygen

Low oxygen concentrations increase larval mortality and hypertrophy of tracheae and other respiratory exchange organs (Greenberg and Ar 1996; Loudon 1988, 1989), and hypoxia conditions (10–10.5% O₂) inhibit growth rate, lead to the development of abnormalities, and to a higher proportion of females in mealworm populations (Loudon 1988). Although development time is similar for both normoxia (21% O₂) and hyperoxia (40% O₂) conditions, hyperoxia induces a lower number of instars, thus resulting in lower final larval biomass than with normoxia (Greenberg and Ar 1996).

Water intake

Although T. molitor can survive under extremely dry conditions for extended periods of time, being able to obtain water from both the food ingested (even substances with low water content) and the atmosphere (Fraenkel and Blewett 1944), larvae grow faster in humid conditions higher than 70% RH. However, the development of mites, fungi, and other microorganisms enhanced by these high-humidity levels are undesirable to the mass rearing of this insect. Mealworms reared on dry substrates exhibit higher growth rates when there is any source of water (Mellandby and French 1958; Oonincx et al. 2015; Urs and Hopkins 1973b). In fact, on low-moisture substrates when metabolic water per unit of food is low (24–35 g water/100 g food), development may halt if there is no drinking water. When suffering from water deprivation, T. molitor larvae ingest less food, and the conversion rate of ingested food into body mass decreases (Murray 1968). The addition of a source of water, such as carrots, results in the increase of survival rates (≥80%) and reduces the development time from 145–151 d to 91–95 d (Oonincx et al. 2015).

The reported effects of water intake on biomass composition are contradictory. Urs and Hopkins (1973) observed that the availability of water increases the concentrations of total lipids while Oonincx et al. (2015) reported that the supplementation of the diets with a water source increases the moisture content but not the total fatty acids content. Both studies found no influence of water on mealworm fatty acid profile.

Protein

Dietary protein and amino acids greatly influence T. molitor life cycle, with direct benefits for larval development time, survival, and weight gain. Supplementation of diets with protein, even in small percentages, has been shown to increase the developmental rate of larvae. Diets supplemented with high protein content (33–39% dry mass) reduce the time for pupation from 191–227 d to 116–144 d at 28°C and 70% RH (Oonincx et al. 2015) and the time to 50% pupation from 95–168 to 79–95 d at 28°C and 65% RH (van Broekhoven et al. 2015), when compared with diets supplemented with low protein content (12% dry mass). Likewise, supplementation with protein increases survival rates from 84–88% to 88–92% (van Broekhoven et al. 2015) and from 19–52% to 67–79% (Oonincx et al. 2015).

The growth rate is enhanced by protein, with a weight gain per larva of 2.3–2.9 mg on a protein-free diet to 45.5–55.6 mg on a diet supplemented with 10 parts of yeast and 90 parts of whole ground wheat during a period of 4 wk (John et al. 1979). Supplementation with casein enhances growth rates from 4.08 g/g of larva with 3% casein to 6.16 g/g of larva with 20% casein (Davis 1970a). The benefits of adding protein to the diet are visible in pupae, with weight gains enhanced from 123 mg/g of food with no supplement to 238 mg/g of food supplemented with protein (Morales-Ramos et al. 2013) and pupal weight from 117–145 mg with a low-protein diet (5% yeast) to 146–161 mg with a rich-protein (40% yeast) diet (van Broekhoven et al. 2015).

Fertility is also highly influenced by the presence of protein in mealworm diet, with increases in average female fecundity from 3 eggs/d in diets without protein increased to 6–7 eggs/d in diets supplemented with protein (Morales-Ramos et al. 2013). Urrejola et al. (2011) also obtained an increase in female fecundity from 5 to 12 eggs/d in diets with yeast at 20% when compared to diets with 2% yeast addition (w/w). Additionally, the period of maximum oviposition occurs earlier in females reared with soybean flour (9th–12th day) than in females reared only with wheat (12th–15th day).

Mealworms seemingly have a highly constrained body protein content with 2–3-fold increases in crude protein content (11.9–39.1% dry mass) of diets resulting in similar body protein composition (van Broekhoven et al. 2015). Yeast, at concentrations of 5–10%, is considered the best protein source acting as a feeding stimulant (Fraenkel 1950; Martin and Hare 1942). Other protein sources that provide optimal effects are 2–32% casein (Davis and Leclercq 1969) and at a lower level, lactalbumin (Davis and Leclercq 1969; Fraenkel 1950; Leclercq 1948). Zein, gliadin, and enzymatic hydrolysates from casein or lactalbumin have residual effects when compared to casein and lactalbumin (Fraenkel 1950). Although soybean is a rich protein source, it contains a potent trypsin inhibitor that negatively influences the larval growth (Birk et al. 1962).

The amino acid contents in the mealworm larval tissues are 8.9–9.9% alanine, 7.9–8.7% aspartic acid, 7.7–8.0% leucine, 6.5–6.8% phenylalanine, 6.5–6.7% valine, 4.6–7.5% proline, 4.6–5.9% arginine, 4.5% isoleucine, 3.9–4.0% threonine, 2.8–2.9% histidine, 1.8–1.9% cysteine, 1.5–1.6% methionine, and 0.7–0.8% tryptophan (John et al. 1979). The ideal diet should contain similar levels of amino acids to those found in larval tissues, with the exception of phenylalanine, which should be provided at 50% of the concentration in body mass, and of the limiting amino acids, threonine and tryptophan, which should be provided at twice the concentration found in larvae tissues.

Fats

Mealworm fat composition is rather constant when fed different diets rich in oleic, linoleic, and palmitic acids (Oonincx et al. 2015). While palmitic and oleic acids remain almost stable, independent of diet, possibly due to the synthesis of fatty acids ad novo by mealworms, linoleic acid may have to be supplied to diets (van Broekhoven et al. 2015). Increased ingestion of polyunsaturated C18 fatty acids lowers the proportion of C18 monounsaturated fatty acids in larval tissues (van Broekhoven et al. 2015).

The addition of lipid to dietary regimes is beneficial at low concentrations while high quantities are unfavorable and potentially pernicious (Morales-Ramos et al. 2013). While cholesterol is a necessary diet ingredient, a fat concentration above 1% does not benefit mealworm life cycle parameters (Fraenkel 1950) and becomes an inhibitory factor at concentrations >3% (Martin and Hare 1942). Moreover, high-fat foods promote the potential agglomeration of the substrate, resulting in lower aeration and movement of mealworms, thus negatively interfering with respiration (Alves et al. 2016). In addition, it has been proven that supplementation with 20% lipid content increases the susceptibility to parasitism by nematodes (Shapiro-Ilan et al. 2008).

Carbohydrates

Tenebrio molitor growth is almost nil in the absence of carbohydrates. The optimal range is 80–85% (Fraenkel 1950). Although Fraenkel (1950) reported no significant differences in the growth of mealworms using glucose or starch, Davis (1974) observed less growth with starch, sucrose, or lactose than with glucose in diets containing amino acid mixtures. Similarly, bacteriological dextrin as a carbohydrate source induced weight gain of almost twice that achieved with glucose (Davis 1974).

The mealworm life cycle is highly influenced by the dietary protein-to-carbohydrate ratio (Martin and Hare 1942; Rho and Lee 2016; Urrejola et al. 2011). Rho and Lee (2016) reported an optimal protein-to-carbohydrate ratio of 1:1 for lifespan and lifetime reproductive success while Martin and Hare (1942) observed maximum growth at a minimum of 50% carbohydrate and a minimum of 15% (or more than 25%) protein in the diet.

The ingestion of diet consisting of several combinations of organic waste material (vegetables), yeast, and Tenebrio excreta results in body protein contents 2-fold higher and body fat content 5- to 6-fold higher than that of food, with a significant reduction in the values of crude fiber and carbohydrates (Ramos-Elorduy et al. 2002). Moreover, mealworms fed on low protein:carbohydrate ratios (0:42 and 7:35) diets have higher body lipid content (Rho and Lee 2014).

Vitamins

Tenebrio molitor shows no growth in the absence of vitamins of the B-complex (carnitine, thiamine, riboflavin, nicotinic acid, pyridoxine, or pantothenic acid) while there is growth, albeit slow, with the lack of biotin or pteroylglutamic acid (Hardouin and Mahoux 2003; Leclercq 1948; Martin and Hare 1942). On the contrary, the addition of vitamins A, C, D, E, and K to the diet provides no beneficial effect (Fraenkel 1950; Martin and Hare 1942). After mealworms reach half- or full-grown size, no addition of vitamins is required for the mealworms to complete larval development and to pupate (Leclercq 1948).