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Introduction
The Dark Winged Fungus Gnat Bradysia impatiens Johannsen is the main pest of technologically advanced forest nurseries in Mexico, although it is also significant in greenhouses for vegetables and ornamental plants (Marín-Cruz et al., 2022; Villanueva-Sánchez et al., 2013). In pine nurseries, mortality rates of up to 30 % have been recorded (Marín-Cruz et al., 2015). Among a range of 17 species of host pines, cespitose species have been the most attacked (Marín-Cruz et al., 2022). Adults and larvae of B. impatiens are vectors of plant pathogens; some of the fungi transmitted include Pythium, Botrytis, Verticillium, Thielaviopsis, Cylindrocladium, Sclerotinia and several species of Fusarium, notably F. circinatum Nirenberg & O´Donnell and F. oxysporum Schltdl. (García-Díaz et al., 2017; Marín-Cruz et al., 2015; Villanueva-Sánchez et al., 2013).
Monitoring methods in nurseries provide information for decision-making regarding the timely application of pest control treatments. Monitoring is useful for determining the population size of a pest, which, when related to impacts and costs, allows defining the level of damage and economic threshold (Onstad et al., 2021). Santos et al. (2012) indicate that to obtain the economic damage level, it is necessary to have parameters on damage per unit of pest and the yield loss of the crop as a function of the total damage.
In pest monitoring, there are several trapping methods that, to be considered effective, should be specific, low-cost, and easy to establish, manage, and evaluate. Shin et al. (2020) demonstrated that yellow sticky traps are efficient for attracting fungus gnats. These are used in several fields of agriculture due to their potential for pest insect control (Shi et al., 2020), as they attract and capture insects due to their color and special adhesive; sometimes, there are variations in color preference depending on the group being studied (Bravo-Portocarrero et al., 2020; Pacheco-Covarrubias et al., 2016; Stukenberg et al., 2018). To be effective, these traps should maintain their color and adhesive stability under natural conditions over time (Barrera et al., 2006); various models and sizes are used, but they are almost always large and difficult to handle (Moravvej et al., 2022; Yoon et al., 2018).
The objectives of this study were to evaluate two types of yellow sticky traps to select the most efficient one for monitoring B. impatiens, to classify population levels according to pest capture and to identify associated organisms, including natural enemies.
Materials and Methods
The research was divided into two stages. In the first stage, the attraction effect of two types of yellow traps and one colorless trap was evaluated. After selecting the best trap, the second stage involved comparing the capture of B. impatiens in four forest nurseries to determine the population levels of the species.
First stage: Evaluation of the attractive effect of traps
At the Atlangatepec nursery in Tlaxcala, located at coordinates 19° 32’ 27.6” N and 98° 10’ 48” W, with an elevation of 2 510 m, maguey Agave salmiana Otto ex Salm-Dyck is produced. In 2022, a target of 500 000 plants was achieved. For its production, plastic trays with 54 cavities of 190 mL were used, with a substrate composed of 25 % pine bark, 25 % peat moss, 25 % perlite, and 25 % vermiculite. This nursery originally produced forest plants; however, currently in Mexico, there are very few nurseries producing temperate climate forest plants, mainly due to productive programs such as 'Sembrando Vida' that require plants with different characteristics; additionally, there was a high percentage of conversion from technified nurseries to nurseries with a traditional system.
The nursery has production modules with metal tables measuring 1.47 m wide, 48.1 m long, and 70 cm high. Each table has a capacity of 384 trays, and tezontle is applied to the soil to prevent weed growth. Production is protected with shade netting and greenhouse plastic. The nursery also produces mezcal agave and fruit trees such as guava and pomegranate.
Types of traps
Yellow circular trap
They were manually constructed using plastic lids from commercial containers. The lids measure 12.1 cm in diameter with a surface area of 114 cm2. One lid served as the base, was spray-painted with acrylic safety yellow paint, allowed to dry, and covered with another lid, but without the rim. The cover was impregnated with motor oil or commercial insect adhesive, whose base is polybutene and aromin. The traps were secured with staples on a wooden stick. Subsequently, traps with captures were stacked on top of each other for storage and later review (Figure 1).
Transparent circular trap
The same type of lids used in the circular trap was used for its preparation. As it is colorless, the trap served as a control to assess the role of the yellow color as an attractant for the Dark Winged Fungus Gnat.
Rectangular Trap (Flag paper trap)
The trap is made of letter-sized polycarbonate (21.59 x 27.94 cm), available in stores selling insecticide products. It has adhesive on the yellow side, is reticulated with lines every 2.4 cm, and is covered with wax paper, allowing for easy peeling off and reattachment. To expedite trap handling, each sheet was cut into four sections, resulting in traps measuring 10.77 x 13.97 cm with an area of 150 cm2; each was stapled onto a stick of the required length according to the plant's height. For installation, the support stick was inserted into the seedling tubes, the wax paper was folded, and it was secured with paper clips. The trap has the advantage that the captured insects are protected when the wax paper is returned to its original position and fixed with the same clips. Insect counting was facilitated by the grid pattern (Figure 2).
Installation of traps
The traps were installed on March 4, 2022, at a maximum height of 10 cm above the apex of the plants; subsequently, they were replaced for three consecutive weeks (March 10, 17, and 24).
The double yellow circular trap was installed with orientations facing south and north (type A and type B, respectively). These orientations were chosen because the main wind gusts come from these directions, which is an important factor in capturing flying arthropods. The transparent circular trap (control) and the yellow rectangular trap (fly paper trap) were placed facing south.
Thirty traps of each type were placed in the maguey beds with a systematic-random design; meaning, the order was chosen randomly, resulting in the placement of flag paper trap, yellow circular, and control. The traps were positioned at a height ≤10 cm from the highest part of the plants (Figure 3), as B. impatiens exhibits a short-flight behavior and hops around the plants and trays (Wilkinson & Daugherty, 1970).
Figure 3 Atlangatepec Nursery, Tlaxcala. A) Larva of Bradysia impatiens and damage on Agave salmiana, B) Yellow circular trap (north and south orientations), C) Control trap (south orientation), D) Flag paper trap (south orientation), E) Systematic arrangement of traps.
The traps were changed weekly (on three dates) and transported to the Forest Entomology Laboratory of the Division of Forest Sciences at the Universidad Autónoma Chapingo. The number of B. impatiens individuals (males and females separately) in each trap was counted using a Leica® stereoscopic microscope and a dissection needle. The species was morphologically identified based on the characteristics described by Villanueva-Sánchez et al. (2013) and Marín-Cruz et al. (2015) before counting. Associated organisms captured were also counted.
Detection of natural enemies of Bradysia impatiens
In March 2022, flies from the Muscidae family were detected. According to Bautista-Martínez et al. (2017), Coenosia attenuata Stein is a predator of B. impatiens. Therefore, from June 2 to June 9, 2023, 20 traps were installed in the nursery to verify the presence of this predator in Tlaxcala.
Statistical Analysis
Primarily, the model was adjusted with Poisson regression to the count data, but evidence of overdispersion was found, as the variance was notably greater than the mean of the data. This limits the suitability of the Poisson model, which requires approximately equal mean and variance measures. This was corrected by using negative binomial regression with a link function, an alternative model that does not demand this criterion.
Counts of captured individuals of B. impatiens were analyzed by fitting generalized linear models using the R program (R Core Team, 2021). Specifically, negative binomial regression was used to model absolute counts, and negative binomial regression for rates was used to model the number of individuals captured per area (cm2). The corresponding link function for the negative binomial regression model is as follows:
where,
µ ij = expected average count of the i-th replication (i = 1, …, 30) of the j-th type of trap
C = constant
T j = effect of the j-th trap type (j = 1, 2, 3, 4)
For the negative binomial regression model for rates, the link function is:
where,
A j = area (cm2) of the j-th trap type
C * = constant
T * j = effect of the j-th trap type
The rate was considered as the total capture of B. impatiens divided by the area (cm2) of the traps. With this adjustment, the error due to differences in trap areas is reduced. To obtain the daily capture of B. impatiens, the weekly capture per trap was divided by the number of days, and the mean per trap type was calculated. Additionally, Tukey's mean comparison was performed to determine statistical differences between trap types with a confidence level of 95 % (P = 0.05).
Second stage: Comparison of population levels of Bradysia impatiens
Based on the results of the first stage, the flag paper trap was selected to determine the population level of B. impatiens in two nurseries in Toluca (“Central” and “Invernadero”) and two in Michoacán (“Morelos” and “Asociación de Silvicultores”). The traps were installed on March 31, 2022, in Toluca and on April 1, 2022, in Michoacán, resulting in two collection dates: April 21 and May 11 for the nurseries in Estado de México, and April 22 and May 12 for the nurseries in Michoacán. These nurseries produce plants of Pinus montezumae Lamb., P. hartweggii Lindl., P. oocarpa Schiede ex Schltdl., P. leiophylla Schiede ex Schltdl. & Cham., P. pseudostrobus Lindl. and P. devoniana Lindl., some of which are cespitose species highly susceptible to B. impatiens attack. However, the species was not considered a significant factor, as all those produced in these nurseries are hosts of the pest, as reported by Marín-Cruz et al. (2015, 2022).
In each nursery, 15 traps were installed for each date, totaling 120 traps evaluated in the four nurseries. These traps were transported to the laboratory, and the captures were counted using the methodology outlined in the first stage. Based on the results, population levels were proposed according to the phytosanitary condition of the nurseries.
Statistical Analysis
A negative binomial regression model was fitted to the count data with the following link function:
where,
µ ij = expected average count in the i-th trap (i = 1, ..., 30) of the j-th nursery
( = constant
V j = effect of the j-th nursery (j = 1, 2, 3, 4)
For the statistical analysis, data from both capture dates were used. To define population levels, the weekly capture was calculated by dividing the raw data by the number of weeks the traps were set up, assuming uniform weekly capture. The captures corresponded to three weeks of exposure; that is, six weeks of evaluation to obtain the two capture dates. Additionally, Tukey's test was applied to the means to identify statistical differences between nurseries (P = 0.05).
Associated Insects
For the first stage, insects were identified at the level of order, family, genus, or species based on their morphological characteristics, preservation condition, and complexity in the trap. For the second stage, organisms from other taxonomic classes within Arthropoda were included. Additionally, dipterans were collected using an entomological aspirator and morphologically identified in the laboratory of the Institute of Epidemiological Diagnosis and Reference (InDRE).
Results
First Stage: Comparison of the Attractiveness Effect of Traps
The number of captured individuals of B. impatiens (Figure 4) and other dipterans of similar size and coloration were recorded. Statistical analysis focused on B. impatiens with a capture percentage of 67.05 % of the total organisms. Of the total pest population, 40.01 % was captured with the flag paper trap, 42.17 % with the yellow circular trap (24.89 % with type B [north orientation] and 22.28 % with type A [south orientation]), and 12.80 % with the control trap (uncolored circular trap).
Figure 4 Bradysia impatiens. A) Female at the base of Agave salmiana, B) Male captured with flag paper trap.
According to Table 1, statistical analysis determined differences in capture between the sticky yellow traps and the control trap; the flag paper trap captured a significantly greater number of insects (P = 0.05). On the other hand, the orientation of the traps (north and south) had no effect, as the capture was similar in both orientations (P = 0.3046). The total number of B. impatiens represents the cumulative capture over the three weeks; similarly, the total represents the cumulative number of captured organisms.
Table 1 Comparison of mean captures of Bradysia impatiens using different types of sticky traps over a three-week evaluation period.
| Type of traps | Weekly captures per trap | Average daily captures per trap |
Total capture of B. impatiens |
Total number of insects |
|---|---|---|---|---|
| Rectangular yellow (flag paper trap) | 160.60 ± 12.11 a | 22.94 | 14 454 | 21 012 |
| Circular yellow (B)1 | 97.11 ± 8.43 b | 13.87 | 8 740 | 13 016 |
| Circular yellow (A)1 | 79.48 ± 6.91 b | 11.35 | 7 153 | 11 651 |
| Circular transparent (control) | 52.33 ± 5.64 c | 7.47 | 4 710 | 6 694 |
1North (B) and south (A) orientations. Means ± standard error with different letters indicate a statistically significant difference according to Tukey's test (P = 0.05).
Second Stage: Comparison of Bradysia impatiens Population Levels
Table 2 indicates that the population levels in the nurseries of Estado de México (“Invernadero” and “Central”) differ from those in Michoacán (“Morelos” and “Asociación de Silvicultores”). Specifically, the “Central” nursery had the highest number of captured fungus gnats, whereas the nurseries in Michoacán had lower counts with no significant differences between them. The average capture per trap reflects the tool's efficacy and varies according to the nursery; the “Central” nursery had the highest average (209 adults over three weeks of evaluation).
Table 2 Capture of Bradysia impatiens by nursery. Data from 30 traps divided into two collection dates, with 15 traps each for three weeks.
| Nursery | Capture per trap | Average daily captures per trap |
Total capture of B. impatiens |
Total number of insects |
|---|---|---|---|---|
| Central | 209.86 ± 38.93 a | 10 | 6 296 | 11 532 |
| Invernadero | 90.90 ± 20.92 b | 4 | 2 727 | 11 274 |
| Morelos | 38.26 ± 2.75 c | 2 | 1 148 | 2 301 |
| Asociación de Silvicultores |
32.96 ± 3.44 c | 2 | 989 | 2 635 |
1Means ± standard error with different letters indicate a statistically significant difference according to Tukey's test (P = 0.05).
On each collection date, the daily capture of B. impatiens (Table 2) was obtained by dividing the three-week capture by the number of days the traps were exposed, and then calculating the average for the two evaluation dates (six weeks in total).
Population Levels of Bradysia impatiens
Population levels were identified as low (<30 adults per week), moderate (30 to 60 adults per week), and high (>60 adults per week). These levels were not associated with economic damage but should be further evaluated in a study relating them to plant damage or mortality (Figure 5).
Associated insects
In the first stage, dipterans from several families were captured, such as: Muscidae (Hunter fly C. attenuata), Ceratopogonidae (Dasyhelea sp.), Chironomidae, Psychodidae, Ephydridae and Syrphidae. In the second stage, individuals of other insects (Hymenoptera, Hemiptera, Coleoptera, and Arachnids) were captured, some unrelated to the fungus gnat, but in the Central nursery, individuals from the families Muscidae (C. attenuata), Psychodidae, and Ceratopogonidae (Dasyhelea sp.) were also found.
From the above, C. attenuata is the only species with economic importance (Figure 6) as a predator of B. impatiens. Moreover, Kleidotoma sp. (Hymenoptera: Figitidae) was captured, which is reported as a parasitoid of B. impatiens and other dipterans, including C. attenuata (Buffington et al., 2020). In the process of detecting natural enemies, it was observed that, out of the total insects (6 327) captured, the flag paper trap captured 1.78 % of C. attenuata (113), 11.49 % (727) of Kleidotoma sp., and 68.92 % (4 361) of B. impatiens.
Figure 6 Coenosia attenuata. A) Adult male, B) female and C) male genitalia in ventral and lateral view.
Coenosia attenuata (Muscidae)
It is a small, light gray fly. Males are smaller than females. The legs are yellowish in males and grayish in females. The abdomen is uniformly light gray in males and has some darker spots in females (Figure 6), and the abdomen has three distinctive transverse black stripes (Kaldor et al., 2022). Bautista-Martínez et al. (2017) reported it for the first time in Mexico, preying on fungus gnats and other pests in Zapopan, Jalisco. The present study is the first report in Tlaxcala and Estado de México, and the second at the national level.
Dasyhelea sp. (Ceratopogonidae)
The biting midges of this genus were identified by M.C. Heron Huerta Jiménez, a Diptera taxonomist at InDRE (Figure 7A). The species belongs to a group with diverse morphology and biology, and they have a cosmopolitan distribution; however, they are not of forest importance (Lu et al., 2020).
Psychodidae
This family exhibits a great variety of feeding habits, some are saprophytes and mycophagous (Figure 7B). Females are occasionally hematophagous, which causes health problems for humans as they are disease vectors, thus they do not have forest importance (Vivero-Gómez et al., 2013).
Chironomidae
They are insects with a wide distribution in bodies of water (Figure 7C), feeding on organic matter and sometimes considered indicators of water contamination (Oviedo-Machado & Reinoso-Flórez, 2018).
Ephydridae
They are a diverse and widely distributed family (Figure 7D). Although they are not significant in forestry, some species are considered agricultural pests (Mathis et al., 2016).
Syrphidae
It is a diverse family found in various habitats (Figure 7E). The subfamily Syrphinae has a large number of species that prey on hemipterans, such as Aphididae, Coccidae, and Aleyrodidae (Arcaya-Sánchez et al., 2017).
Kleidotoma sp.
The females (Figure 8) actively seek dipteran larvae in environments such as animal feces and decomposing organic matter. Therefore, there is a high probability of Kleidotoma sp. parasitizing larvae of B. impatiens (Fontal & Nieves-Aldrey, 2004); however, the hosts of most species are unknown. While it can play an important role as a parasitoid of the pest, it has also been reported as a parasite of C. attenuata (Buffington et al., 2020).
General capture of flag paper trap
In the “Atlangatepec” nursery, the average capture of B. impatiens using flag paper trap was 68.78 % (14 454 adults out of a total capture of 21 012 organisms) over three weeks (30 traps per date, with a total of 90 traps). In the “Morelos” nursery, the average capture of B. impatiens was 49.89 % (1 148 out of 2 301), and in the nursery “Asociación de Silvicultores”, it was 37.53 % (989 out of 2 635). In the “Central” and “Invernadero” nurseries, it was 54.59 % (6 296 out of 11 532) and 24.18 % (2 727 out of 11 274), respectively. These percentages correspond to six accumulated weeks (30 traps per nursery) over the two collection dates.
The flag paper trap captured, on average per day, 14 males and 11 females in Atlangatepec; one male and one female in the Morelos and Asociación de Silvicultores nurseries; seven males and three females in the central nursery; and three males and one female in the Invernadero nursery.
Discussion
In the transparent traps, there was lower capture than in the yellow ones, indicating the influence of this color as an attractant for adult B. impatiens, which is consistent with findings by An et al. (2019), Atakan and Pehlivan (2015), Murtaza et al. (2019), Prema et al. (2018), and Allan et al. (2020).
In the comparison of trap types, one of the factors considered was orientation. In the study area (Atlangatepec, Tlaxcala), the main wind gusts come from the south and north. Therefore, yellow circular traps (A and B) were chosen for placement; however, wind predominantly occurred from south to north, so flag paper and control traps were placed facing south. The results did not show statistical differences between orientations, although numerically, there was higher capture in the north-facing trap. This could be due to the wind possibly repelling the presence of B. impatiens, as indicated by Wilkinson and Daugherty (1970) that adults have weak flight. Based on these results, it is recommended to place traps in orientations where there is not excessive wind for species monitoring.
The study used relatively small circular (114 cm2) and rectangular (150 cm2) traps. Despite their size, when there were higher insect captures, counting became challenging, so samples were taken from the surfaces. For the circular trap, pie-shaped sections were sampled, and for the rectangular ones, rows from the grid were taken. Generally, in other studies, monitoring traps are large and difficult to evaluate, and in some cases, they are similar in size to those tested in this study. Sun et al. (2017) suggest using artificial vision algorithms, which allow for automatic counting and classification of insects to optimize time. However, this technology requires extensive training, so manual counting remains the most common method for evaluating yellow sticky traps.
The population levels classified as low, medium, and high were chosen arbitrarily based on the phytosanitary condition of the nurseries, so they are reference values that can be adjusted with the quantification of damage by B. impatiens and the costs of control measures. These levels could be used as a basis for making decisions about the population size of the pest. With this estimation, it is suggested to initiate preventive activities when the population level reaches the medium level, such as: management of ambient humidity and substrate; removal of the circular moss cover generated on the substrate surface, commonly called 'cookie', as it is a conducive space for larvae to proliferate; and the application of control methods as indicated by the Insecticide Resistance Action Committee (IRAC, 2024).
The spread of B. impatiens in the country may be facilitated by the transport of infested plants (Marín-Cruz et al., 2022), which in turn leads to the movement of C. attenuata. This species has now been found in Estado de México and Tlaxcala. According to Zou et al. (2021), C. attenuata can consume 12 to 24 adults of B. impatiens per day. However, Kleidotoma sp. has the potential to parasitize many dipteran species and may even be reducing the population of the predatory fly C. attenuata, thus reducing its predatory effectiveness (Buffington et al., 2020).
The flies Dasyhelea sp. and those from the families Chironomidae and Ephydridae were captured using yellow sticky traps. The resemblance of these flies in size, color, and shape to B. impatiens can confuse nursery workers when counting captures, highlighting the need for training nursery staff in the identification of adults. Moreover, flies from the families Psychodidae and Syrphidae can also confuse evaluators and lead to incorrect decisions (Arcaya-Sánchez et al., 2017; Vivero-Gómez et al., 2013).
Conclusions
The flag paper trap can be useful for monitoring the dark-winged fungus gnat Bradysia impatiens in forest nurseries, as it captures more adults than circular traps. Population levels need to be precisely determined in future studies to establish the economic threshold of the pest based on control costs and damage quantification. The predatory fly C. attenuata, which preys on B. impatiens, has a low capture rate (1.78 %), indicating that the traps are not a threat to their populations. The capture of associated organisms is significant compared to B. impatiens, and due to their similar appearance and size, they can be easily confused. An integrated pest management plan for B. impatiens is necessary, incorporating the flag paper trap and the predator C. attenuata as biological control. Additionally, Kleidotoma sp. should be studied to identify its potential as a parasitoid of both the pest and C. attenuata.
