Host-Seeking and Acceptance Behaviour of Plodia interpunctella (Lepidoptera: Pyralidae) Larvae in Response to Volatile Compounds Emitted by Amaranth

Abstract

In this study, the seeking behaviour and food acceptance of larvae of Plodia interpunctella Hübner (Lepidoptera: Pyralidae) were analysed under laboratory conditions. Larval orientation and feeding preferences were assessed using a selection arena for neonate larvae and a four-way olfactometer for third-instar larvae. Stimulants included amaranth bars with additives (honey and chocolate) and natural amaranth (toasted grain only). The results showed that amaranth volatiles influence the orientation and feeding behaviour of this polyphagous insect. A marked preference for sugar-rich foods was observed, with amaranth with honey and amaranth with chocolate being the food sources most frequently chosen by the neonate larvae. These individuals exhibited a gregarious feeding behaviour and did not engage in cannibalism. The third-instar larvae also showed a preference for sweet food but were more attracted to the amaranth–additive combination. In the four-way olfactometer bioassays, chocolate was the most frequently chosen stimulus, while cellophane did not differ significantly from air. An analysis of volatile compounds by gas chromatography mass spectrometry (GC-MS) revealed that amaranth with chocolate releases more volatile compounds (16) compared with honey (12) and natural amaranth (6), suggesting that these volatiles could possibly influence the larvae’s choice of food source.

1. Introduction

Plodia interpunctella Hübner (Lepidoptera: Pyralidae) is a cosmopolitan pest of stored products. This insect has fully adapted to the human environment and continues to expand its host range, even in regions exposed to extreme climatic conditions, such as Antarctica [1]. Thus, it is considered the most significant pest of processed foodstuffs and stored grain [2,3,4]. In the Mexican state of Morelos, in 2020, P. interpunctella was identified as the cause of damage in amaranth warehouses [5], leading to the description of its biological cycle and biotic injury potential in the three amaranth products with the highest infestations [4]. This caused the loss of more than 50% of the stored amaranth bars, mainly chocolate amaranth bars, honey amaranth bars, and natural amaranth bars (toasted grain only).

Several authors have documented that host selection is significantly influenced by host quality and the specific volatile profile of the host [6,7,8,9]. It has also been reported that the high content of total sugars in food is a powerful stimulant and directly influences the feeding behaviour of P. interpunctella larvae by positively affecting host seeking and acceptance [4,6,8,10,11]. Plodia interpunctella has the ability to survive and complete its life cycle in all amaranth products; however, this species benefits from amaranth bars that contain additives such as honey and chocolate, as they increase its reproductive potential and life expectancy, attributable to the combination of grain proteins with the sugar content of the additives [4,6,12].

Previous studies have demonstrated that this insect pest can detect the wrappings of certain foods and pierce packaging primarily composed of cellophane and polypropylene, thereby gaining access to food items that have already undergone processing and packaging for retail sale [8,13,14,15]. The volatile profile of toasted amaranth contains more than one hundred compounds from different classes, with hexane acid and acetic acid being the most abundant in natural amaranth [16]. Furthermore, studies have mentioned that the treatment and preparation of amaranth for consumption affect its volatile composition [15,16].

In the larval stages of P. interpunctella, host-seeking and acceptance behaviour for feeding has not been reported so far; therefore, this study investigates the larval behaviour in relation to stored amaranth products and identifies the volatile compounds emitted from each evaluated product that can serve as larval attractants. The identification of potential attractants will contribute to the development of an effective tool for the ethological management of this pest.

2. Materials and Methods

2.1. Insects and Amaranth

For each bioassay, neonate and third-instar larvae obtained from the insect hatchery of the Insect Ecoethology Laboratory of the Jicarero Higher School of Studies, belonging to the Autonomous University of the State of Morelos (EESJ-UAEM), were used under the following conditions: 25 ± 2 °C, 65% ± 5% relative humidity, and a photoperiod of 12:12 h L:D. The insect brood stock was obtained from contaminated bars in the warehouses and continued to be fed with a mixture of amaranth varieties. The insects used belonged to the third generation of insects once the quarantine was over. The amaranth used in the bioassays was obtained from amaranth grain warehouses located in Temoac, Morelos, Mexico. Amaranth energy bars with chocolate (made from cocoa, cinnamon, and sugar), amaranth energy bars with honey (made from panela molasses from refined cane sugar), and natural amaranth energy bars (made only with roasted grain) were selected for the bioassays, as these products have been reported to have the highest larval contamination, with losses exceeding 50% of the stored products. These bars are part of Mexican culture and traditions and are commonly marketed under the name “Alegrias”.

2.2. Dispersion and Feeding-Preference Bioassays of Neonate Larvae

For the dispersion bioassays, Petri dishes with a diameter of 10 cm and a height of 2 cm were used as selection arenas, under conditions of 25 ± 2 °C, 65% ± 5% relative humidity, and a photoperiod of 12:12 h L:D. Three types of amaranth bars were evaluated in this study: amaranth with chocolate, amaranth with honey, and natural amaranth. Thirty newly hatched neonate larvae were released in the centre of the arena, and ten replicates of the bioassay were performed (N = 300, experimental unit = 30 larvae, n = 10). A piece of 1 g of each type of amaranth to be evaluated was placed equidistantly into the Petri dishes. Each repetition comprised two controls. The first control consisted of food with the same weight and position as the bioassay food, with the aim of evaluating whether the food lost weight naturally on its own. The second control consisted of placing only the larvae in the arena under the same conditions as the bioassay larvae, but without food, with the aim of determining the weight of the larvae and ascertaining whether it remained constant or decreased.

The behaviour of the larvae was observed over a period of 300 s, and the time taken to disperse was recorded. The larvae were then left to consume whichever food they selected for a period of 24 h. A video camera was attached to the apparatus to monitor and confirm the larvae’s fidelity to the food. Subsequently, the food was weighed once more to record the mean consumption. The larva was also weighed to assess whether it had experienced a significant weight gain after feeding. At the conclusion of the experiment, both the food and control larvae were weighed. The weight of the sample was measured using a high-precision analytical balance (Accuris Instrument model W3200-5K, Minnetonka, MN, USA, 2018) [17].

2.3. Bioassay on the Feeding Preference of Third-Instar Larvae to Amaranth Volatiles

For this bioassay, a four-way olfactometer with a diameter of 15 cm was utilised under the same conditions as described for the previous bioassay. In this bioassay, 40 third-instar larvae were utilised, having fasted for a duration of 24 h. The treatments evaluated consisted of amaranth with chocolate, amaranth with honey, natural amaranth, and the controls, specifically the chocolate that had been used for the preparation of the bars, honey (molasses), cellophane (from the packaging), and a stream of pure air. The larvae were exposed to the controls to ascertain their preference.

In each bioassay, air was delivered by an oxygen pump (AQAPURA model K1590 2.5 W, Monterrey, N.L., Mexico) at a flow rate of 200 mL/min, regulated by a flowmeter (SHLLJ, model LZQ 1 LPM, Jiangsu Province, Huaian, China). The air passed through an activated carbon filter, which removed impurities, and then through a water trap (an Erlenmeyer flask containing 200 mL of distilled water), which restored humidity. The air then entered four glass chambers (10 cm high and 15 cm inner diameter), each containing 16 g of one of the three amaranth bars studied in this bioassay (amaranth with chocolate, honey, and natural), and one of the controls (chocolate, honey, air, and cellophane) was placed in the fourth chamber. Volatile compounds emitted by the odour sources were transported to the larvae, which were released into the centre of the olfactometer. After every five repetitions, the olfactometer and the amaranth-containing chambers were cleaned with a stream of clean air for 300 s; then, the olfactometer was rotated to avoid bias in data collection or so that some other factor, such as position or shade, would not influence the insect’s behaviour. Larvae behaviour was observed for 300 s.

The data that were recorded included the food items selected, as well as the duration of exploration and food source selection. The exploration time was defined as the time between the release of the larva until its introduction into one of the olfactometer arms, while the selection time was defined as the time it took the larva to traverse from the entrance of the arm to the volatile-emitting source. The term “food acceptance” was used to denote the moment at which the larva arrived at the food source and initiated the ingestion process. Behavioural transition matrices and corresponding ethograms were created based on the frequency of behaviour observed in the larvae.

2.4. Production of Ethograms

The elaboration of ethograms was based on the observations made during the four-way olfactometer food selection tests. Preliminary observations enabled the sequence of behavioural events to be determined. This sequence comprised the following: random walk, tracking, detection, oriented walk, return, and food acceptance.

To ascertain whether the behavioural acts of the insect were sequenced in an orderly fashion or occurred randomly, first-order Markov transition matrices were constructed using the frequency of observed behaviours. The corresponding ethograms were made for each bioassay with its respective control and the comparison of controls [18,19,20,21].

To calculate the expected frequency (FE), the following formula was implemented:

FE = (∑rows × ∑columns)/total.

To calculate transition, the following formula was implemented:

Transition = (FO/∑total) × 100.

To calculate χ², the following formula was implemented:

χ² = (FO − FE)²/FE,

where FO = observed frequency and FE = expected frequency.

The individual transitions with an observed frequency greater than the expected frequency were considered significantly greater than random, if calculated χ² < tabulated χ² (p = 0.05) [22].

2.5. Extraction and Identification of Volatile Compounds

The extraction of volatile compounds was carried out on 50 g of each amaranth bar and the packaging of 10 bars. The experiment was conducted within a glass chamber, the dimensions of which were 10 cm in height and 15 cm in inner diameter. The chamber was equipped with two outlets. An activated carbon filter was positioned at the air inlet to remove impurities. A Pasteur pipette containing 125 mg of Super Q (SQ) 80/100 adsorbent material (Alltech Assoc. Inc., Chicago, IL, USA) was positioned at the chamber outlet and connected to a vacuum pump (Welch, USA) with a flow rate of 0.5 L/min, which was regulated by a flowmeter (Cole, Palmer, Ev-03217-06, USA). The extraction of volatiles was carried out for a period of 4 h at a temperature of 26 ± 2 °C and a relative humidity of 60% ± 5 RH. Compounds attached to the Super Q were eluted with 1 mL of hexane. The eluate was then reconcentrated with a stream of nitrogen gas until it reached a volume of 300 µL. The compound was stored at 4 °C until use.

The separation and identification of volatile compounds was carried out using a gas chromatograph coupled to a mass spectrometer (GC-MS) (Agilent, 7890-5975C, Santa Clara, CA, USA), equipped with an SLB-5MS capillary column (30 m × 0.25 mm × 0.25 µm) (Sigma Aldrich Supelco, Darmstadt, Germany). Hydrogen was utilised as the carrier gas, with a constant flow rate of 2 mL/min. The initial oven temperature was set at 40 °C for a period of two minutes, with a subsequent increase of 10 °C per minute until a temperature of 200 °C was attained. This was followed by an increase of 20 °C per minute until it reached a temperature of 250 °C, which was maintained for a duration of 10 min. Two microlitres (n = 8) of each extract were injected, with the injector operated in splitless mode for a period of 2 min at a temperature of 260 °C [16].

As a control, an eluate was injected from the pipette with SQ to extract the volatiles from the empty chamber. The selection of volatile compounds of interest was based on the presence of peaks in at least seven of the eight injections per type of amaranth bar, with an abundance greater than 90% being deemed significant. The selected volatile compounds were identified by retention time, and their mass spectra were compared with those of the spectral library (NIST/EPA/NIH). In addition, the Kovats retention index [23] was calculated for each compound, with reference standards obtained from Sigma Aldrich (Toluca, Mexico).

2.6. Statistical Analysis

The software programme Sigma Plot 12.0. (Systat Software Inc., Düsseldorf, Germany) was utilised for the analysis of the data. To determine the selection frequency of neonate larvae, the results of the bioassays in the selection arena were analysed using a one-way analysis of variance (ANOVA) and Tukey’s analysis of separation of means, p < 0.05. Inequality in their distribution was evaluated with the Lorenz curve and the Gini concentration index (GI) [24] (Equation (1)). The classification of the concentration of neonate larvae after dispersal, measured through the Gini index, was carried out using the scale presented in

Table 1. Classification of inequality using the Gini index.

Gini Index Value	Inequality
0.101–0.250	Null to low
0.251–0.500	Low to medium
0.501–0.700	Medium to high
0.701–0.900	High to very high
0.901–1	Very high to absolute

.

$$I_G=\sum _{i=1}^{n-1}\left(P_i-Q_i\right)/\sum _{i=1}^{n-1}pi$$

(1)

where

• P = Accumulated proportion of amaranth.
• Q = Accumulated proportion of neonate larvae in the amaranth.
• n = Types of amaranth bars in which larvae are distributed.

To determine the food preference of third-instar larvae, an analysis of the results of the bioassays conducted in the four-way olfactometer was carried out using a chi-squared goodness-of-fit test (χ²) to compare observed and expected frequencies, and if there was a significant difference at p ≤ 0.05, comparisons between treatment pairs were performed using a chi-square of independence test (χ²). A one-way analysis of variance (ANOVA) followed by a Tukey test was used to compare mean response times in each bioassay with the control. To analyse the abundance of the volatile compounds identified in each type of amaranth bar evaluated in the behavioural bioassays, the area under the curve (AUC) of each compound was calculated and then compared via the one-way analysis of variance (ANOVA) test. This procedure was only carried out where compounds were present in all three of the extracts. In instances where the compound was present in two extracts, a Student’s t-test was used, and when the compound was present in only one type of bar, the mean was reported.

3. Results

3.1. Dispersion and Food Preference of Neonate Larvae of P. interpunctella

Regarding food preference, 43.1% of the neonatal larvae expressed a preference for amaranth with chocolate, 39.76% for amaranth with honey, and 17.14% preferred natural toasted amaranth, with a significant difference between amaranth with chocolate and amaranth with honey vs. natural toasted amaranth: F_2,14 = 117.478; 14; p < 0.050; n = 10; N = 300 (Figure 1A). Amaranth with chocolate was selected most frequently, but the mean dispersion time of the larvae was lower when compared with larvae that chose amaranth with honey. The dispersion time was also significantly higher for natural amaranth, with means of 25.21, 25.91, and 158.75 s, respectively. Furthermore, the dispersion curve is indicative of the propensity of populations to select sweeter foods, such as amaranth combined with chocolate or honey. The calculation of the index G = 0.33 reveals that the dispersion inequality is low, with a tendency towards the median when tabulated in the table. To be precise, the distribution of insects across the three foods is not uniform, and there is a tendency for the insects to prefer amaranth with sweet additives (Figure 1B).

The neonate larva of P. interpunctella used in choice tests experienced a mean weight gain of 2.133 mg, while the control larva presented a mean weight loss of 7.1 mg. A significant difference in weight was observed when comparing before and after the experiment (Figure 2).

3.2. Preference of Amaranth Volatiles by Third-Instar Larvae

There was a response from all forty larvae when chocolate was utilised as a control. The preferred food was amaranth with honey, followed by amaranth with chocolate. There was no significant difference observed between natural amaranth and the control χ² = 9.6, df = 3, p = 0.002, n = 40 (Figure 3 and Figure 4).

With honey as a control, there was no response from three larvae; in this case, the amaranth samples with chocolate and those with honey were selected most, with equal frequencies, followed by natural amaranth and the control groups, between which there were no significant differences (F = 6.567, df = 3, p = 0.087, n = 37).

With air as a control, thirty-nine out of forty larvae responded to the stimuli. The most frequently selected food was amaranth with honey, followed by amaranth with chocolate (χ² = 29, df = 3, p = 0.001, n = 39). The least attractive treatments for the larvae were natural amaranth and air, with no significant difference between these two (Figure 3 and Figure 4).

For cellophane as a control, there was no response from four larvae, and no significant difference was detected between amaranth with honey and amaranth with chocolate or between amaranth with honey and natural amaranth. A significant difference was observed between amaranth with chocolate versus natural amaranth and the control χ² = 4.44, df = 3, p = 0.217, n = 36 (Figure 3 and Figure 4).

A comparison of controls between honey, chocolate, cellophane, and air was also performed, with chocolate and honey as the most significantly selected controls (χ² = 13.7895, df = 3, p = 0.05, n = 38), but without any significant difference between the two, followed by cellophane and air. In this case, significant differences were observed between the latter two controls. Significant differences were also observed between cellophane, honey, and chocolate (Figure 3 and Figure 4).

In the analysis of the percentage of larvae that accepted the food, taking into consideration the various controls, it was determined that, in the case of chocolate as a control, the larvae demonstrated a significant preference for amaranth with honey, followed by amaranth with chocolate, and finally natural amaranth, with no significant difference observed between the latter two treatments (χ² = 9.6, df = 3, p = 0.002, n = 4) (Figure 3).

In the case of the honey control, the larvae demonstrated a significant preference for amaranth with honey and amaranth with chocolate, followed by natural amaranth, and finally the control (χ² = 6.567, df = 3, p < 0.05, n = 37). No significant difference was observed between amaranth with honey and amaranth with chocolate (Figure 3).

For cellophane as the control, the larvae demonstrated a clear preference for amaranth combined with honey and amaranth combined with chocolate, with natural amaranth and the control following in second and third positions, respectively (χ² = 4.44, df = 3, p = 0.217, n = 46). No significant difference was observed between amaranth with honey and amaranth with chocolate, nor between natural amaranth and the control group.

In the context of the air control, the larvae exhibited a marked preference for amaranth with honey, a secondary preference for amaranth with chocolate, and finally a preference for natural amaranth and the control treatment. No significant differences were observed between the latter two treatments (χ² = 29, df = 3, p < 0.001, n = 39) (Figure 3).

Finally, when comparing the response times of the larvae when selecting a food source, a significantly faster response was exhibited when chocolate was used as a control compared to the other treatments (F_3,79 = 16.889, p < 0.05, n = 8; Figure 3).

3.3. Identification of Volatile Compounds from Amaranth Products

A total of twenty-three volatile compounds were identified in the amaranth products, including seven important compounds specific to cellophane, six compounds in natural amaranth, twelve compounds in amaranth with honey, and sixteen compounds in amaranth with chocolate (see

Table 2. Volatile compounds identified in amaranth products and cellophane.

Compound	Formula	Molecular Weight (g/mol)	Class	CAS	Retention Time	Kovax Retention Index (IR)	Area Under the Curve ± SEM				Statistical Test
							ACH	AH	NA	Cellophane
Acetic acid	C2H4O2	60.052	Carboxylic acid	64-19-7	3	948	156,285 ± 2100	219,453 ± 4679	-	-	t = −1.233; gl = 1, 12 p = 0.25,1 n = 7
Benzaldehyde	C7H6O	106.12	Aldehyde	100-52-7	3.5	1106	1,354,195 ± 376	1,684,596 ± 7908	-	-	t = −1.616; gl = 1, 12 p = 0.713, n = 7
3,5-Octadien-2-ol	C8H14O	126.2	Alcohol	69668-82-2	4.2	1119	1,218,130 ± 2312	-	-	-	Mean average ± SEM
5-Octen-2-ona	C8H14O	126.19	Ketone	22610-86-2	4.7	1125	-	-	-	1,437,611 ± 2312	Mean average ± SEM
1,3-dimethyl-5-ethylbenzene	C10H14	134.22	Aromatic hydrocarbon	934-74-7	5	1158	-	-	-	643,165 ± 3019	Mean average ± SEM
D-limonene	C10H16	136.24	Alkene	138-86-3	5.6	1159	6,073,353 ± 126	5,100,216 ± 1189	4,173,709 ± 8398	-	F = 0.729; gl = 2, 23; p = 0.494, n = 8
Alpha pinene	C10H16	136.23	Terpene	136.23	6.1	1163	421,043 ± 760	303,622 ± 88,710	-	-	t = 1.005; gl = 2, 14; p = 0.332, n = 8
Nonanal	C9H118O	142.23	Aldehyde	124-19-6	6.4	1167	901,788 ± 3918	-	-	-	Mean average ± SEM
Undecane	C10H8	156.31	Alkane	1120-21-4	6.9	1168	-	-	-	111,456 ± 193	Mean average ± SEM
Decanal	C10H20O	156.27	Aldehyde	112-31-2	7.4	1170	340,420.5 ± 8315 *	9,666,011 ± 155	6,960,869 ± 5015	-	F = 3.856; gl = 2, 23; p = 0.037, n = 8
Nonanoic acid	C9H18O2	158.23	Carboxylic acid	112-05-0	7.9	1182	1,820,397 ± 1847	415,710 ± 5851	312,033 ± 44,037	-	F = 153.67;gl = 2, 23; p = 0.09, n = 8
Decanoic acid	C10H20O2	172.26	Carboxylic acid	334-48-5	8.7	1194	1,756,537 ± 18,397	-	-	-	Mean average ± SEM
Dodecanoic acid	C12H24O2	200.32	Carboxylic acid	143-07-7	9.5	1628	1,369,119 ± 38,178	-	-	-	Mean average ± SEM
Tetradecanal	C14H28O	212.37	Aldehyde	124-25-4	10.3	1716	299,004 ± 326	334,994 ± 97,157	5,744,056 ± 9459 *	-	F = 3.455; gl = 2, 23; p = 0.05, n = 8
Pentadecane	C15H32	212.42	Alkene	629-62-9	11.2	1734	537,295 ± 4436	454,644 ± 1108	-	-	t = 0.692; gl = 1, 12; p = 0.502, n = 7
Butylated hydroxytoluene	C15H24O	220.34	Alkene	128-37-0	12	1771	-	-	-	760,333 ± 1047	Mean average ± SEM
n-pentadecanol	C15H32O	228.41	Alcohol	629-76-5	12.7	1936	236,545 ± 3418	218,689 ± 2602	559,092 ± 107,347 *	-	F = 8.171; gl = 2, 23; p = 0.431, n = 8
Octadecane	C18H38	254.4	Alkane	593-45-3	14.3	1940	-	-	-	352,882 ± 318	Mean average ± SEM
Hexadecanoic acid	C16H32O2	256.43	Carboxylic acid	57-10-3	15	1982	1,480,218 ± 7469	985,217 ± 8912	-	-	t = −2.084; gl = 1, 14; p = 0.056, n = 7
Eicosano	C20H42	282.56	Alkane	112-95-8	15.8	2073	227,177 ± 7493	206,587 ± 3187	242,836 ± 5033	-	F = 0.108; gl = 2, 20; p = 0.890, n = 7
Heneicosane	C21H44	296.583	Alkane	629-94-7	16.8	2108	167,200 ± 2654	194,269 ± 3358	-	-	t = −0.633; gl = 1, 14; p = 0.537, n = 8
Cyclopentaxylsane, decamethy	C10H30O5	370.77	Siloxane	208-764-9	17.9	2202	-	-	-	1,178,222 ± 174	Mean average ± SEM
Hexanedioic acid1,6-bis(2-ethylhexyl)ester	C22H42O4	370.57	Ester	103-23-1	19.5	2462	-	-	-	766,032 ± 10,004	Mean average ± SEM

Where: ACH = Amaranth with chocolate; AH = Amaranth with honey; NA = Natural amaranth; The symbol * denotes significantly different compound.

).

Decanal was one of the compounds found in all three types of amaranth evaluated. It was most abundant in amaranth with honey and natural amaranth, at levels significantly higher than in amaranth with chocolate (F_2,23 = 3856; p < 0.05; n = 8). In chocolate amaranth, the most abundant compound was nonanoic acid. This compound is also found in all three types of amaranth, but it is significantly less abundant in natural amaranth (F_2,23 = 153.67; p < 0.050; n = 8) (Table 2).

D-Limonene is another compound found in all three types of amaranth and is significantly more abundant in amaranth with chocolate and amaranth with honey (F_2,23 = 0.729; p < 0.5; n = 8). α-Pinene, acetic acid, and benzaldehyde are compounds found in both amaranth with chocolate and amaranth with honey, with no significant difference between them. These compounds were not found in the volatile profile of natural amaranth (Table 2).

Tetradecanal, n-pentadecanol, and icosano are compounds that are found in the profiles of three types of amaranth, with icosano (F_2,23 = 8.171; p < 0.05; n = 8) and tetradecanal (F_2,23 = 3.455; p < 0.05; n = 8) being significantly more abundant than in natural amaranth.

4. Discussion

Since P. interpunctella is a generalist insect, it can feed on more than one food source and select the food that best suits its development, seeking food rich in micro- and macronutrients, sugars, and moisture [25].

In this case, the dispersal and foraging behaviour of the neonate larvae was geared towards the search for food with a higher sugar content. It is known that the larvae are adapted to select a food source that provides them with sufficient energy to survive this vulnerable stage of development, which is associated with a high mortality rate. Furthermore, the type of food they choose will determine the duration of their biological development cycle [26,27].

The neonatal larvae in this study showed a preference for foods with a high sugar content: amaranth with chocolate and amaranth with honey. Similar results have been documented in the neonatal larvae of Manduca sexta (Linnaeus) (Sphingidae) [28]; Choristoneura fumiferana (Clemens) (Tortricidae) [29]; Gnorimoschema operculella (Zeller) (Gelechiidae) [30]; Ostrinia nubilalis (Hübner) (Crambidae) [31]; Uraba lugens (Swartz) (Nolidae) [32]; Heliothis zea (Boddie) (Noctuidae) [33]; Spodoptera littoralis (Boisduval) (Noctuidae) [30]; and Trichoplusia ni (Hübner) (Noctuidae) [30], which, when offered, were attracted to sucrose, fructose, glucose, maltose, or any other type of sugar, accompanied by increased feeding rates.

The physical characteristics of food play a decisive role in determining the choice of individual larvae. Food that is very hard, liquid, or presents a sticky texture is typically refused, as they prefer to feed on chewable and crumbly food, such as flours, bland grains, and smooth food [26,34].

In the context of restricted environments and diets of varying quality, insects develop mechanisms to compensate for nutritional deficiencies. For instance, they may increase their intake of sugary foods to increase their weight and size [35,36,37].

The third-instar larvae also preferred amaranth bars with chocolate or honey; however, significant differences were observed when responses to chocolate or honey without amaranth were compared with those to amaranth bars with chocolate or honey, respectively. Consequently, it can be inferred that the synergy between the grain and the sugar contained in the chocolate and honey is the key factor that attracts the larvae [38].

Understanding the behavioural patterns that allow pests to successfully find food and cause damage is important if we are to prevent the spread of contamination [39]. For this reason, ethograms are of paramount importance in this area of research, as they facilitate the identification of the factors that determine food-seeking and food acceptance behaviour [40].

Undoubtedly, the presence of volatile compounds is essential for orientation and food selection. This is clearly reflected in the ethograms of P. interpunctella that show that the larvae are immediately activated once they encounter the volatiles released within the system, and that their search for and orientation towards food is determined by the type of volatiles that are most attractive to them.

In a study on the attraction of the red flour beetle Tribolium castaneum (Herbst) to wheat flour, it was observed that the orientation of the insect was determined not only by the concentration of moisture present during the release of volatile compounds but also by the nutritional condition of the individual insect. Thus, an insect that had grown up on a more nutritious diet did not show an interest in flour, whereas an insect with limited and poor food sources immediately moved towards the flour [41].

In the case of P. interpunctella larvae, the main conditions that provoke an increase in attraction are the presence of grain, a sweet additive, such as sugar and/or honey, and the absence of packaging. The latter facilitates the release of volatile compounds from the food that perfectly guide the larva to its target. Silhacek et al. (2003) [40] mention that the release of volatile compounds in a ventilated and aerated warehouse can also contribute to pest control. The compounds become mixed together, which results in the pest becoming confused and disoriented and unable to find its preferred food source, thus remaining where it was oviposited.

Several studies mention that the volatile compounds of some grains, such as peanuts, corn, and wheat, elicit a good orientation and reach response in third-stage P. interpunctella larvae. However, they also state that the entire process is contingent on the concentration of total sugars, since the presence of these directly influences the feeding preference of the larvae and that, regardless of the proteins and fats in the food, it is the synergy with sugar that is decisive when determining which food source is the optimum host [6,10,42,43].

Similarly, in the case of adults of Chillo partellus (Swinhoe), table sugar and sucrose were the best phagostimulants in warehouse trapping systems, in combination with a Bacillus thuringiensis treatment in a trap-and-kill (attracticide) trapping system [44].

However, there are some cases, such as that of the larvae of the codling moth Cydia pomonella (Linnaeus) (Tortricidae), in which an increased sugar concentration had a detrimental effect, as an increase in the sugar concentration in apple substrates drastically reduced consumption and delayed the onset of feeding. In the silkworm Bombix mori (Linnaeus), the effect of various sugars and compounds related to larval feeding was also tested, and it was observed that glucose was a weak stimulant for this insect [7].

In general, the response of Lepidoptera in the larval stage to sugar concentration is uncertain. In most cases, the response is positive with a strong attraction. However, further research on the role of host volatiles in behavioural bioassays is essential, as there are some cases where sugar concentration can result in the opposite effect.

Among the compounds identified in amaranth bars, nonanal is particularly noteworthy. It has previously been reported as a potent attractant for P. interpunctella in wheat bars [45,46,47,48]. Moreover, the substance has been utilised in combination with the sex pheromone ZETA and nonanoic acid in pilot trapping systems in chocolate and dog food factories in Europe [25]. Nonanoic acid has also been identified in the volatile profiles of amaranth bars, and some authors refer to it as a classic volatile compound of natural grains and sugars, as it is a fatty acid and has a very vinegar-like odour [16,27,49].

In addition, several authors have mentioned that pyralid insects are sensitive and display a flight response in the presence of acetic acid, isoamyl alcohol, and α-pinene compounds, which are also found in the volatile compounds of amaranth [27,38].

5. Conclusions

In conclusion, the larvae of P. interpunctella demonstrate a sustained preference for amaranth-based products containing sweet additives, a behaviour likely guided by volatile compounds present in both the food and its packaging. This sensory-driven response suggests that such compounds play a key role in host selection and feeding behaviour. The elevated attraction to chocolate- and honey-flavoured bars highlights their potential as effective lures. These insights support the development of ethological control strategies—such as attractant-based traps or host confusion systems—which are already being implemented in storage facilities in Morelos, Mexico.