Effect of Larval Density...

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Effect of Larval Density on Food Utilization Efficiency of
Tenebrio molitor (Coleoptera: Tenebrionidae)
Author(s): Juan A. Morales-Ramos and M. Guadalupe Rojas
Source: Journal of Economic Entomology, 108(5):2259-2267.
Published By: Entomological Society of America
URL: http://www.bioone.org/doi/full/10.1093/jee/tov208
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ECOLOGY
AND
BEHAVIOR
Effect of Larval Density on Food Utilization Efficiency
of Tenebrio molitor (Coleoptera: Tenebrionidae)
JUAN A. MORALES-RAMOS1 AND M. GUADALUPE ROJAS
USDA-ARS National Biological Control Laboratory, Biological Control of Pests Research Unit, Stoneville, MS 38776.
J. Econ. Entomol. 108(5): 2259–2267 (2015); DOI: 10.1093/jee/tov208
ABSTRACT Crowding conditions of larvae may have a significant impact on commercial production
efficiency of some insects, such as Tenebrio molitor L. (Coleoptera: Tenebrionidae). Although larval densities are known to affect developmental time and growth in T. molitor, no reports were found on the
effects of crowding on food utilization. The effect of larval density on food utilization efficiency of T. molitor larvae was studied by measuring efficiency of ingested food conversion (ECI), efficiency of digested
food conversion (EDC), and mg of larval weight gain per gram of food consumed (LWGpFC) at increasing larval densities (12, 24, 36, 48, 50, 62, 74, and 96 larvae per dm2) over four consecutive 3-wk periods.
Individual larval weight gain and food consumption were negatively impacted by larval density. Similarly,
ECI, ECD, and LWGpFC were negatively impacted by larval density. Larval ageing, measured as four
consecutive 3-wk periods, significantly and independently impacted ECI, ECD, and LWGpFC in a negative way. General linear model analysis showed that age had a higher impact than density on food utilization parameters of T. molitor larvae. Larval growth was determined to be responsible for the age effects,
as measurements of larval mass density (in grams of larvae per dm2) had a significant impact on food utilization parameters across ages and density treatments (in number of larvae per dm2). The importance of
mass versus numbers per unit of area as measurements of larval density and the implications of negative
effects of density on food utilization for insect biomass production are discussed.
KEY WORDS yellow mealworm, food conversion, rearing, biomass production, insects for food
The yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae), is produced in large numbers and
sold in the United States for a variety of purposes. The
larvae of T. molitor are one of the most common foods
for captive mammals, birds, reptiles, and amphibians
because they are easy to propagate, harvest, and feed
(Martin et al. 1976, Barker et al. 1998, Finke 2002).
The existence of this type of world-wide industry has
inspired organizations like FAO to propose insects as a
potential source of food and feed (van Huis et al.
2013). T. molitor has been proposed as a candidate to
produce insect biomass as a source for animal feed
(Ramos-Elorduy et al. 2002, van Huis et al. 2013, Makkar et al. 2014, Sánchez-Muros et al. 2014), for aquaculture (Ng et al. 2001, Riddick 2014, Barroso et al.
2014), and for human consumption (DeFoliart 1992,
1999; Ramos-Elorduy 1997, 2009; Gahukar 2011; van
Huis et al. 2013; Shockley and Dossey 2014). Insects
are a promising source of high-quality animal protein
with a substantially lower ecological footprint than vertebrate livestock (Finke 2002, 2013; Oonincx et al.
2010; Oonincx and de Boer 2012; van Huis et al. 2013;
Shockley and Dossey 2014). The topic of the potential
for insects to contribute to sustainable human food security has received the attention of several
1
Corresponding author, e-mail address: Juan.m[email protected]
.usda.gov.
organizations. The UN Food and Agricultural Organization (UN FAO) have proposed a program of feeding
people with alternative food sources including insects
(Gahukar 2011).
Commercial availability of insect biomass (e.g.
T. molitor) also provides an opportunity for developing
new technologies for mass production of biological control agents. The potential of T. molitor as factitious prey
for insect predators has been explored (Saint-Cyr and
Cloutier 1996, De Clercq et al. 1998, Grundy et al.
2000, Costello et al. 2002, Lemos et al. 2003, Pappas
et al. 2007, De Bortoli 2011). Another application is as
a host for in vivo mass production of entomopathogenic
nematodes (Shapiro-Ilan et al. 2002, 2008, 2012). The
low level of technology required for T. molitor production makes it ideal for the small biological control industry. In addition, T. molitor extracts could be used to
supplement artificial diets for entomophagous arthropods (Morales-Ramos et al. 2014).
The use of commercially produced T. molitor to produce insect biomass for food and to aid the production
of biological control agents will depend on lowering the
costs of its production. Research efforts to reduce the
cost of producing T. molitor have focused on optimizing
reproduction and reducing labor (Morales-Ramos et al.
2011a), improving adult rearing conditions (MoralesRamos et al. 2012), and improving diets (MoralesRamos et al. 2010, 2011b, 2013). However, rearing
conditions of immature insects, particularly the density
Published by Oxford University Press on behalf of Entomological Society of America 2015.
This work is written by US Government employees and is in the public domain in the US.
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JOURNAL OF ECONOMIC ENTOMOLOGY
of larvae, may also have a significant impact on production efficiency. Increased larval densities delayed or inhibited pupation in many tenebrionid species including
T. molitor (Tschinkel and Willson 1971). Weaver and
McFarlane (1990) reported a significant and negative
impact of larval density on pupal mass in T. molitor,
suggesting reduction in growth rates at increasing densities. Increasing larval densities in Gnatocerus cornutus (F.) (Coleoptera: Tenebrionidae) significantly
delayed development and increased mortality and cannibalism (Savvidou and Bell 1994). Similarly, development time increased and pupal weight was reduced as
larval densities increased in Alphitobius diaperinus
Panzer (Coleoptera: Tenebrionidae) (Parween and
Begum 2001). Larval crowding arrested pupation in
Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) (Botella and Ménsua 1986). A similar phenomenon was observed in T. freemani (Hinton), which failed
to pupate for more than 6 months under crowded conditions (Nakakita 1982). Hirashima et al. (1995) also
observed a delay in the release of ecdysone in T. freemani larvae under crowded conditions.
Although larval densities affected developmental
time and growth in some tenebrionids, no reports were
found on the effects of crowding on food utilization.
Negative effects of crowding on food conversion efficiency would impact productivity in a commercial colony of T. molitor. Existing production methods tend to
favor high larval densities in T. molitor commercial production facilities in order to reduce space demands
(J. A. M.-R. unpublished data). The objective of this
study was to determine if increasing larval densities
have negative effects on the food utilization parameters
and therefore biomass production of T. molitor.
Materials and Methods
Colony Maintenance. The T. molitor colony used
in this study was originally established in 2005 from
stock donated by Southeastern Insectaries Inc. (Perry,
GA), and it has been continuously grown at the
National Biological Control Laboratory (Stoneville,
MS) for the past 9 yr. The rearing methods were as
described by Morales-Ramos et al. (2012) using stacked
fiberglass trays (MFG Tray Co., Linesville, PA) with
screened bottoms (500-mm pore size) to grow the larvae, boxes with screened bottoms (850-mm pore size) to
hold the adults, and collection of first instars in a second tray at the bottom. The colony was fed mostly with
wheat bran (>90%) and supplemented with 5–10% dry
potato squares once a week. Adults were misted with
water twice a week with a spray bottle. Because larvae
of T. molitor have the ability to take up water dissolved
in subsaturated air (Dunbar and Winston 1975) the relative humidity in the rearing room was maintained at
70% or higher. The room was kept under dark conditions at 26 C.
Experimental Design. To create different larval
density conditions, increasing numbers of larvae
were placed into modified stacked Petri dishes (20 by
60 mm diam.) separated by a nylon screen standard
number 45 (350-mm openings) as described by
Vol. 108, no. 5
Morales-Ramos et al. (2013). The screen allowed frass
particles to fall to the second dish at the bottom while
keeping the larvae and food in place. Larval densities
were measured as number of larvae per square decimeter (dm2) as described by Morales-Ramos et al.
(2012). The experimental dishes had a bottom area of
2733.97 mm2 equivalent to 0.2734 dm2. Eight treatments consisting of 12, 24, 36, 48, 60, 72, 84, and 96
larvae per dish were created, which corresponded to
densities of 44, 88, 132, 176, 220, 263, 307, and 351
larvae per dm2, respectively (Table 1).
Four-week-old larvae from the stock colony were
separated according to sizes by using a series of sieves
from standard number 20 to number 35. Larvae passing through sieve number 30 (600 mm) and remaining
with sieve number 35 (500 mm) were selected for the
experiment. This portion included larvae from the
fourth to the sixth instar based on their head capsule
measurements (Morales-Ramos et al. 2014). Larvae
sorted by this method were used to create 10 groups of
larvae for each of the eight density treatments consisting of the appropriate number of larvae for each of the
treatments. Each group of larvae was weighed at the
beginning of the experiment, and their initial weight
was recorded. Groups were transferred to dishes as
described above and were provided with food consisting of 80% wheat bran and 20% of a supplement composed of 17:2:1 parts of dry potato, dry egg whites, and
soy protein, respectively. The quantity of the food provided varied according to the larval density and was
equivalent to 34 mg of food per larvae per dish
(Table 1). All dishes were transferred to an environmental chamber where they were maintained at 27 C,
75% RH, and under darkness for a 3-wk period.
At the end of the 3 wk, larvae from each dish were
separated from the food and weighed as a group.
Remaining food and frass were dried in a vacuum oven
at 50 C and negative pressure of 1,000 hPa for 24 h
and weighed. Live weight gain (LWG) was determined
by subtracting the initial larval weight from the accumulated weight of live larvae from each dish. Dry
weight gained (DWG) was calculated by multiplying
LWG by the dry weight proportion of T. molitor larvae
(0.38) as reported by Finke (2002). Food consumed
(FC) was calculated by subtracting the weight of the
remaining food from the weight of the food provided.
Food consumption and weight gained data were used
to calculate food utilization parameters as described by
Waldbauer (1968). Food assimilated (FA) was
Table 1. Density treatments for groups of T. molitor larvae
Treatment
number
1
2
3
4
5
6
7
8
Larvae
per dish
Larvae
per dm2
Initial
food (mg)
12
24
36
48
60
72
84
96
44
88
132
176
220
263
307
351
400
800
1200
1600
2000
2400
2800
3200
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MORALES-RAMOS AND ROJAS: EFFECT OF LARVAL DENSITY IN T. molitor
calculated by subtracting the frass weight from the
weight of the food consumed (FA ¼ FC – frass).
Weight of food converted was equal to the dry weight
gain (DWG). Efficiency of ingested food conversion
(ECI) was calculated as ECI ¼ DWG * 100 / FC and
efficiency of digested food conversion (ECD) was calculated as ECD ¼ DWG * 100 / FA (Waldbauer 1968).
Additionally, live weight (in mg) gained per gram of
food consumed (LWGpFC) was calculated as LWG /
(FC * 0.001).
After data had been recorded for the first 3-wk
period, larvae were returned to their corresponding
dishes, provided with additional food mix (84 mg per
larvae), and returned to the environmental chambers
for another 3-wk period. At the end of the second 3-wk
period, larvae groups were processed as described
above and data was recorded. This process was
repeated for another 2, 3-wk periods completing a total
of 4, 3-wk periods of data collection. Food provided
per larvae increased every 3-wk period as larvae food
consumption increased with age. Dishes were monitored daily for food consumption to prevent larvae
from running out of food. If food was totally consumed,
new food was dried, weighed, added, and recorded.
Food utilization parameters were also calculated for
the full 12-wk experimental period.
Data Analysis. Food utilization parameters at each
of the 4, 3-wk periods and the full 12-wk period were
analyzed using ANOVA; means were compared among
treatments and control by Tukey–Kramer’s HSD test
(SAS Institute 2013a). The parameters ECD and ECI,
expressed as percentages, were arcsine converted for
analysis (Zar 1999). Linear regression was used to analyze the impact of larval density on the values of food
utilization parameters. General linear model was used
to analyze and compare the impact of larval density
and 3-wk period (larval age) and their interaction on
food utilization parameters (SAS Institute 2013b).
Density was also calculated as larval mass per dm2.
The mean larval mass per dm2 during a given 3-wk
period was calculated as (initial weight þ ending
weight)/2 for each of the observations. Linear regression was used to analyze the impact of larval mass density on food utilization parameters. A multiple linear
regression model was used to analyze the relative
impact of both types of measurements of density (larval
numbers and larval mass per unit of area) on the food
utilization parameters.
Results
Analyses of the whole 12-wk data showed that larval
density had a significant impact on individual larval
weight gain (F ¼ 71.91, df1 ¼ 7, df2 ¼ 72, P < 0.0001),
food consumed per larvae (F ¼ 15.55, df1 ¼ 7, df2 ¼ 72,
P < 0.0001), live weight gained per food consumed
(LWGpFC; F ¼ 79.57, df1 ¼ 7, df2 ¼ 72, P < 0.0001),
efficiency of digested food conversion (ECD;
F ¼ 49.15, df1 ¼ 7, df2 ¼ 72, P < 0.0001), and efficiency
of ingested food conversion (ECI; F ¼ 76.63, df1 ¼ 7,
df2 ¼ 72, P < 0.0001; Table 2). All the food utilization
parameters showed a significant linear correlation with
2261
larval density. Individual larval live weight gain
and food consumed per larvae were negatively
impacted by larval density (b ¼ 0.144. R2 ¼ 0.86,
F ¼ 498.27, df ¼ 78, P < 0.0001 and b ¼ 0.305,
R2 ¼ 0.52, F ¼84.23, df ¼ 78, P < 0.0001, respectively;
Fig. 1). The parameter based on live weight LWGpFC
was impacted negatively (b ¼ 0.147, R2 ¼ 0.84,
F ¼ 404.19, df ¼ 78, P < 0.0001) by larval density,
respectively (Fig. 2A). The parameters ECD and ECI
were both impacted negatively by larval density
(b ¼ 0.03, R2 ¼ 0.799, df ¼ 78, P < 0.0001 and
b ¼ 0.015, R2 ¼ 0.84, df ¼ 78, P < 0.0001, respectively; Fig. 2B and C).
Comparison of food utilization parameters among
different 3-wk periods revealed significant impact of
larval age on food utilization efficiency. Not surprisingly, larval weight gain and food consumption per larvae increased significantly and progressively from one
3-wk period to the next (b ¼ 4.78, R2 ¼ 0.887,
F ¼ 2497.7, df ¼ 318, P < 0.001, and b ¼ 27.32,
R2 ¼ 0.969, F ¼ 9815.56, df ¼ 318, P < 0.0001, respectively). As larvae grew, food consumption and weight
gain were expected to increase proportionally. More
surprising was the negative effect of larval growth on
the rest of the food utilization parameters (Tables 3–5).
Larval biomass produced per g of food (LWGpFC) and
ECI were negatively impacted by larval aging
(b ¼ 8.3, R2 ¼ 0.643, F ¼ 572.27, df ¼ 318, P < 0.0001
and b ¼ 0.0032, R2 ¼ 0.63, F ¼ 542.5, df ¼ 318,
P < 0.001, respectively). The impact of larval aging on
ECD was significant (F ¼ 83.2, df1 ¼ 3, df2 ¼ 316,
P < 0.0001), but it was nonlinear and the highest value
was observed during the second 3-wk period (Table 4).
A general linear model analysis on the impact of
larval density treatment and the 3-wk period (larval
age) on food utilization parameters revealed that larval
aging had a greater impact than the density treatments
on these parameters (Table 6). Although density in larvae per dm2 remained constant within treatment and 3wk period, density measured as larval mass per dm2
increased within density treatments as larvae aged and
grew, and the relative space available to larvae diminished. Linear regression analyses on food utilization
parameters of mixed ages and treatments (all pooled
data) versus larval mass density expressed in g/dm2
showed a significant linear fit for LWGpFC
(R2 ¼ 0.692, F ¼ 713.84, df ¼ 318, P < 0.0001), dryweight ECD (R2 ¼ 0.474, F ¼ 286.71, df ¼ 318,
P < 0.0001), and dry-weight ECI (R2 ¼ 0.71,
F ¼ 777.81, df ¼ 318, P < 0.0001; Fig. 3). Similar analyses of pooled data using number of larvae per dm2 as
the independent variable did not show significant linear
fit, with a lack of fit for F ¼ 3.05, 4.05, and 3.33 for
LWGpFC, ECD, and ECI, respectively; and lack of fit
for P values smaller than 0.0001. This indicates that
larval mass is more relevant than the number of larvae
as a measure of density and food utilization parameters
were more affected by larval mass density than by the
number of larvae per area unit.
However, multiple linear regression analysis of a
model including both measurements of density (as
number of larvae and as mass) as independent variables
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Vol. 108, no. 5
Table 2. Food utilization parameters of T. molitor larvae at eight different larval densities during a 12-wk period
Treatment
1
2
3
4
5
6
7
8
LWG (mg)
DWG (mg)
FC (mg)
LWGpFC (mg)
ECD (%)
ECI (%)
136.2 6 6.5a
132.1 6 5.0ab
126.0 6 6.2b
117.5 6 6.6c
111.7 6 2.4cd
103.8 6 5.2de
97.5 6 6.6e
96.4 6 6.2e
51.8 6 2.5a
50.2 6 1.9ab
47.9 6 2.3b
44.6 6 2.5c
42.5 6 0.9cd
39.4 6 2.0de
37.0 6 2.5e
36.6 6 2.4e
601.3 6 42.0a
619.4 6 28.3a
616.2 6 28.9a
583.3 6 26.6ab
583.3 6 14.5ab
554.1 6 22.4bc
534.5 6 30.7c
530.1 6 22.3c
227.0 6 9.3a
213.3 6 4.7b
204.6 6 6.5c
201.3 6 3.2c
191.6 6 2.5d
187.3 6 4.7de
182.3 6 6.0e
181.7 6 5.8e
20.29 6 0.50a
19.44 6 0.39b
18.84 6 0.78bc
18.28 6 0.68cd
17.94 6 0.29de
17.50 6 0.44ef
16.95 6 0.40f
17.03 6 0.55f
9.39 6 0.37a
8.82 6 0.20b
8.49 6 0.27c
8.35 6 0.12c
7.97 6 0.10d
7.79 6 0.19de
7.58 6 0.25e
7.56 6 0.24e
Individual Larval Weight Gained (mg)
LWG, live larvae weight gain; DWG, dry weight gain of larvae; FC, food consumed; LWGpFC, live larvae weight gained per g of food consumed; ECD, dry weight efficiency of digested food conversion; ECI, dry weight efficiency of ingested food conversion.
Mean 6 SD. Means with the same letter within columns are not significantly different after Tukey–Kramer HSD test at a ¼ 0.05.
150
A
140
130
120
110
100
90
80
70
0
50
100
150
200
250
300
350
400
Food Consumed per Larva (mg)
700
B
650
600
550
500
450
0
50
100
150
200
250
300
350
400
Larval Density per dm2
Fig. 1. Effect of larval density expressed as larvae per dm2 on live weight gained of individual larvae (A) and food
consumed per larvae (B) within 12-wk period. Dots represent observations and lines represent linear regression models. (A)
Model: Y ¼ 143.23 – 0.144X, R2 ¼ 0.865, F ¼ 498.27. (B) Model: Y ¼ 637.27 – 0.305X, R2 ¼ 0.519, F ¼ 84.23.
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MORALES-RAMOS AND ROJAS: EFFECT OF LARVAL DENSITY IN T. molitor
2263
260
A
LWGpFC (mg)
240
220
200
180
160
B
ECD (%)
22
20
18
16
14
C
10
ECI (%)
9
8
7
6
0
50
100
150
200
250
300
350
400
2
Larval Density per dm
Fig. 2. Effect of larval density expressed as larvae per dm2 on live weight gained per gram of food consumed (LWGpFC)
(A), efficiency of digested food conversion (dry-weight ECD) (B), and efficiency of ingested food conversion (dry-weight ECI)
(C) within 12-wk period. Dots represent observations and lines represent linear regression models. (A) Model: Y ¼ 227.22 –
0.147X, R2 ¼ 0.838, F ¼ 404.19. (B) Model: Y ¼ 54.539 – 0.0304X, R2 ¼ 0.799, F ¼ 309.71. (C) Model: Y ¼ 22.722 – 0.0147X,
R2 ¼ 0.838, F ¼ 404.19.
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Table 3. Live weight (biomass) gained per g of food (wet weight) consumed in mg (LWGpFC) of T. molitor larvae at eight different densities obtained during 4, 3-wk periods
Density treatment
Three-week period
First
1
2
3
4
5
6
7
8
299.5 6 19.3a
276.4 6 11.6a
247.9 6 20.4a
255.8 6 20.3a
247.1 6 10.7a
243.7 6 8.7a
241.2 6 16.3a
227.1 6 16.2a
Second
Third
Fourth
248.1 6 18.5b
233.6 6 11.2b
235.6 6 6.0a
233.9 6 14.7b
225.9 6 10.4b
221.4 6 10.5b
207.9 6 16.9b
217.8 6 14.6a
227.4 6 7.3c
217.4 6 9.3c
216.3 6 5.4b
204.1 6 16.2c
191.6 6 8.1c
191.0 6 8.3c
188.5 6 8.8c
178.5 6 8.2b
214.8 6 13.8c
197.3 6 9.6d
180.3 6 12.7c
184.3 6 11.1d
171.8 6 8.0d
167.0 6 8.9d
161.6 6 13.0d
167.3 6 10.3b
Mean 6 SD. Means with the same letter within rows are not significantly different after Tukey–Kramer HSD test at a ¼ 0.05.
Table 4. Percent efficiency of digested food conversion (ECD) of T. molitor larvae at eight different densities obtained during 4, 3-wk
periods
Density treatment
Three-week period
First
1
2
3
4
5
6
7
8
20.22 6 1.18b
20.15 6 0.64b
19.83 6 0.74b
17.93 6 1.93b
19.05 6 0.47b
17.81 6 0.47b
17.63 6 0.73b
17.47 6 0.73b
Second
Third
Fourth
21.91 6 0.480a
21.70 6 0.42a
21.38 6 0.20a
20.84 6 0.11a
20.19 6 0.31a
19.96 6 0.42a
18.93 6 0.64a
19.03 6 0.62a
20.53 6 0.32b
20.21 6 0.43b
19.90 6 0.46b
18.59 6 2.13b
18.44 6 0.35c
18.09 6 0.58b
17.52 6 0.37b
17.15 6 0.49b
19.60 6 0.98b
17.98 6 0.80c
16.98 6 1.38c
17.31 6 0.77b
16.43 6 0.61d
16.06 6 0.71c
15.56 6 0.81c
16.12 6 0.86c
Mean 6 SD. Means with the same letter within rows are not significantly different after Tukey–Kramer HSD test at a ¼ 0.05.
Table 5. Percent efficiency of ingested food conversion (ECI) of T. molitor larvae at eight different densities obtained during 4, 3-wk
periods
Density treatment
Three-week period
First
1
2
3
4
5
6
7
8
12.00 6 0.67a
11.22 6 0.37a
10.13 6 0.82a
10.31 6 0.74a
10.12 6 0.38a
9.85 6 0.29a
9.73 6 0.64a
9.19 6 0.65a
Second
Third
Fourth
10.26 6 0.71b
9.71 6 0.42b
9.76 6 0.20a
9.71 6 0.55a
9.34 6 0.38b
9.22 6 0.42b
8.66 6 0.65b
9.05 6 0.55a
9.44 6 0.27c
9.05 6 0.37c
9.00 6 0.22b
8.49 6 0.65b
8.02 6 0.30c
7.96 6 0.34c
7.85 6 0.34c
7.45 6 0.32b
8.89 6 0.59c
8.11 6 0.41d
7.49 6 0.52c
7.65 6 0.45c
7.14 6 0.32d
6.95 6 0.36d
6.73 6 0.54d
6.97 6 0.42b
Mean 6 SD. Means with the same letter within rows are not significantly different after Tukey–Kramer HSD test at a ¼ 0.05.
Table 6. General linear model (GLM) analysis results of larval
density treatment, 3-wk period, and their interaction effect on
food utilization parameters
Parameter
R2
F
Larval
density
F ratio
3-Wk
period
F ratio
Interaction
F ratio
LWGpFC
ECD
ECI
0.866
0.825
0.867
67.52
43.73
67.9
70.41
82.08
75.65
511.63
241.84
503.59
3.12
2.64
3.07
Models df1 ¼ 31, df2 ¼ 288, P < 0.0001. All partial effects were significant with P < 0.0002.
LWGpFC, live larvae weight gained per g of food consumed; ECD,
dry weight efficiency of digested food conversion; ECI, dry weight
efficiency of ingested food conversion.
showed significant impact of both measurements on
dependent variables LWGpFC, ECD, and ECI. The
t values for parameter null hypothesis of independent
variable “number of larvae per dm2” were 2.55,
8.29, and 3.1, with P ¼ 0.011, P < 0.0001, and
P ¼ 0.0021 for LWGpFC, ECD, and ECI, respectively.
The t values for parameter null hypothesis of independent variable “larvae mass in g/dm2” were 23.29,
13.33, and 24.24, for dependent variables
LWGpFC, ECD, and ECI, respectively, with
P < 0.0001 in all cases.
Discussion
Food utilization parameters of T. molitor were significantly and negatively impacted by increasing larval
densities. However, regression analyses showed a stronger impact of larval age than larval densities on food
utilization parameters of T. molitor. The density of
larval mass increases with age even if larval numbers
per unit of area remained constant. This was confirmed
by the significant effect of larval mass density
October 2015
MORALES-RAMOS AND ROJAS: EFFECT OF LARVAL DENSITY IN T. molitor
2265
350
A
LWGpFC (mg)
300
250
200
150
100
24
B
ECD (%)
22
20
18
16
14
12
C
14
ECI (%)
12
10
8
6
4
0
5
10
15
20
25
30
Larval Mass Density in g / dm2
Fig. 3. Regression analyses of all 4, 3-wk periods showing the effect of larval mass density expressed as grams of larvae
per dm2 on live weight gained per gram of food consumed (LWGpFC) (A), efficiency of digested food conversion (dry-weight
ECD) (B), and efficiency of ingested food conversion (dry-weight ECI) (C) within 3-wk period. Circles represent observations
and lines represent linear regression models. (A) Model: Y ¼ 246.167 – 3.904X, R2 ¼ 0.692, F ¼ 713.84. (B) Model: Y ¼ 0.201 –
0.00173X, R2 ¼ 0.474, F ¼ 286.71. (C) Model: Y ¼ 0.041 – 0.00153X, R2 ¼ 0.71, F ¼ 777.81.
(in mg/dm2) on food utilization parameters across ages
and treatments (Fig. 3). This implies that the space
available between larvae is the factor that determines
larval density effects and therefore larval densities
increase as larvae grow in a space of constant dimensions. Tschinkel and Willson (1971) determined that
the negative effects of larval crowding in some tenebrionids are due to physical contact (mechanical
2266
JOURNAL OF ECONOMIC ENTOMOLOGY
stimulation) among larvae. Although mechanical stimulation effects were relatively weak in T. molitor, pupation was delayed significantly by physically stimulating
larvae (by rubbing them with chains) and applying
vibration at low larval densities (Tschinkel and Willson
1971). However, multiple regression analysis of our
data showed significant impact of both, number of larvae per area unit and mass of larvae per area unit, on
LWGpFC, ECD, and ECI parameters. This may mean
that the effects of density are not just caused by
mechanical contact, but by other factors related to
number of individuals, which could be chemical in
nature.
Weaver and McFarlane (1990) hypothesized that
reduction of growth due to increased larval density in
T. molitor was due to reduced feeding opportunity
induced by conspecific competition. Our study shows
that individual food consumption was significantly
reduced as larval density increased. Although this
seems to support Weaver and McFarlane (1990)
hypothesis, it is more likely that reduction in growth
was due to the combination of reduced ingestion and
the observed reduction in ECD and ECI in response
to increased larval density. This effect may be a behavioral response to the frequency of conspecific contacts
as Tschinkel and Willson (1971) demonstrated. However, our results could not rule out the sublethal effects
of high temperatures due to metabolic heat production
as a potential factor in reducing food utilization efficiency. Metabolic heat has been reported as a major
problem in insect colonies where rearing conditions
often result in extremely high larval densities (Howell
and Clift 1972, Tanaka et al. 1972). Larval aggregations
of some dipterans, such as Calliphora vomitoria (L.),
can produce from 5 to 27 C temperature elevations
from ambient temperature (Turner and Howard 1992).
High larval densities of late instars of T. molitor can
increase temperature by 5 to 10 C inside rearing trays
depending of the level of density (J. A. M.-R. unpublished data). Separating the effects of high larval densities from metabolic induced high temperature effects
is a difficult task and will be the focus of future
research.
Results of this study may have important implications for improving T. molitor rearing conditions and
for increasing T. molitor biomass production. Although
space considerations in arthropod mass rearing are
important in reducing production costs, crowding larvae to save space may be counterproductive. The
research presented herein demonstrates that increasing
larval density (in numbers or mass) impacts productivity, reducing efficiency of food conversion linearly. This
may result in higher food expenses and lower biomass
production. More research is needed to determine the
optimal larval density to balance space and productivity
in T. molitor mass production operations.
Acknowledgments
We acknowledge the technical assistance of Mr. Matthew
McDaniel in the maintenence of the stock colony.
Vol. 108, no. 5
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Received 22 April 2015; accepted 27 June 2015.
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