Introduction

Encarsia formosa Gahan (Hymenoptera: Aphelinidae) is an important natural enemy used for the control of whiteflies Trialeurodes vaporariorum Westwood and Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), which are major pests of Solanaceae and Fabaceae crops. Encarsia formosa is considered a cosmopolitan species because it is widely used in several countries in biological control programs under greenhouse conditions (van Lenteren 2012).

In Colombia, the supply of this natural enemy for biological control programs is limited. Van Lenteren & Bueno (2003) reviewed the state of commercial augmentative biological control in the country and found that most of the efforts have focused on the production of Trichogramma species for sugarcane pests. Although research on E. formosa has been ongoing in Colombia since the 1980s (Español & Corredor 1988, Tello et al2007, Aragón et al2008, de Vis & van Lenteren 2008), a successful commercial mass rearing process that can offer E. formosa to growers has not yet been established. For mass production, one of the problems is the labor cost for scouting populations, because it is a time-demanding activity that increases the production cost (Sørensen et al2012).

The number of insects used and produced during the mass rearing process is usually unknown because counting each insect takes a considerable amount of time. This problem could be solved by estimating the total number of insects in a production batch using sampling methods (Pedigo & Buntin 1994), but determining which method to use depends on the insect population parameters. Few studies have estimated insect population sizes in the E. formosa mass rearing process, and those studies have only reported the quantity of E. formosa at the end of the process (Scopes 1969, Scopes & Biggerstaff 1971). No studies have determined the density of whitefly nymphs or the population of E. formosa pupae during the process. In fact, over the past 5 years, only one study aimed to develop indirect methods to measure populations in an insect mass rearing; however, this study avoided using direct counts (Zheng et al2015).

Therefore, this study aims to adapt a method for quantifying populations of insects in the mass rearing of E. formosa. Specifically, as part of this method, we estimated (i) the T. vaporariorum population density that occurs in a production batch, using the leaf area of the host plant as a base, (ii) the minimum representative sample unit to accurately estimate the density of E. formosa pupae, and (iii) the relationship between the number of E. formosa adults released and the total number of individuals produced in a production batch.

Material and Methods

The study was conducted in a plastic film greenhouse (L, 8 × W, 6.4 × H, 3.8 m) with roof ventilation located on the Nueva Granada Military University Campus in Cajicá, Cundinamarca, Colombia (4°56′49.1″N 74°00′50.8″W), at 2.558 m above sea level. The mean annual temperature inside the greenhouse was 17.07 ± 0.11°C, the maximum temperature was 30.17 ± 0.29°C, and the minimum temperature was 10.09 ± 0.085°C. The mean relative humidity was 72.94 ± 0.38%, and the photoperiod was 12:12 during the experiment, which used natural light. The temperature and relative humidity were not controlled because the facility aims to produce insects that are acclimated to commercial greenhouses.

Production scheme for mass rearing E. formosa on common bean plants using T. vaporariorum as a host

The mass rearing production scheme of E. formosa was based on one reported in the literature (Scopes 1969, van Lenteren & Tommasini 2003) and was modified to achieve a weekly batch scheme production. Each batch consists of 30 pots with 90 plants of common bean Phaseolus vulgaris L. (Fabales: Fabaceae) as described below.

Three common bean plants (cultivar ICA-Cerinza) were grown in 1-kg pots using soil and rice husk as substrates. The apical growth of the plants was limited by cutting off the first shoot of trifoliate leaves, and only the primary leaves remained. Each week, new leaves were removed to ensure that only the primary leaves were presented. At the sixth week of development, plants were put inside an insect cage (L, 120 × W, 80 × H, 80 cm). Next, whitefly adults were collected from a separated mass rearing greenhouse using an ecoVAC® Insect vacuum (ecoTech, Bonn, Germany) and released inside the cage were the plants were held during 24 h. Approximately 2000 adults of T. vaporariorum per plant were released to ensure that the total area under the leaf was covered. After the 24 h, adults were removed using the insect vacuum and taken back to the separate rearing greenhouse in order to stop egg-laying.

Subsequently, the plants with T. vaporariorum eggs were maintained in the cage until nymphs reached the third instar. A × 40 magnifying lens was used to count nymphs and determine the instar using morphological characteristics. Once the third instar stage was reached, adults of E. formosa were released as follows.

Previously to the release, E. formosa pupae from old batches were collected and kept in a dark cage with glass jars embedded in its walls. Recently emerged (24 h) E. formosa adults were collected from the glass jars using a mouth-operated insect aspirator. Then, the E. formosa adults were released inside cages with infested plants by opening the container of the aspirator upside down and gently hitting in the base. The number of wasps released was established using the reported relationship of one wasp for each 17 nymphs (Aragón et al2008). Six weeks after the release of E. formosa, the leaves with E. formosa black pupae were harvested and placed in a dark cage to obtain E. formosa adults.

Determination of T. vaporariorum population density on the plant

In this study, population density was determined using the number of third instar nymphs of T. vaporariorum on the leaf (nymphs/cm2). This parameter was used to estimate rapidly and reliably the total number of nymphs on a leaf without counting the total number of nymphs. To determine the dispersion of this parameter, 30 leaves with T. vaporariorum third instar nymphs were collected from different production batches of the mass rearing. Each leaf was analyzed in the laboratory; the total number of nymphs on the leaf was counted using a stereomicroscope, and the leaf area (cm2) was measured using ImageJ® software (ver. 1.46a) (Abramoff et al2004).

The recorded data were used to adjust a multiple linear regression model using the statistical package R (R Core Team 2018). The dependent variable was the total number of nymphs per leaf, and the explanatory variables were the leaf area and the density of third instar nymphs.

To accurately estimate the mean of the explanatory variables, the minimum sample size for each variable was estimated. The sample size was calculated using the standard deviation and the mean of each explanatory variable, as shown in Eq. 1. This equation is based on a binomial negative distribution that is appropriate for species with an aggregated population, as in the case of whiteflies.

$$ n=\frac{1}{\frac{\overline{X}+1/\widehat{k}}{D^2}} $$
(1)

where n is the minimum sample size for each explanatory variable (i.e., leaf area or density), X is the sample mean, D is the significance level (0.05), and k is the binomial negative distribution parameter that is calculated using Eq. 2.

$$ \widehat{k}=\frac{\overline{X}}{s^2-\overline{X}} $$
(2)

where s2 is the sample variance. K values less than 1 represent a population with an aggregated distribution. The data were plotted in an effort curve using the sample size on the x-axis and the mean and standard deviation (bars) for each sample size on the y-axis. The minimum sample unit was established as the value of the x-axis where the mean and standard deviation values stabilized (Kranz 1988).

Determination of the minimum sample size of E. formosa pupae for three different sample units

Three different sample units and their corresponding sample sizes were evaluated for the quantification of E. formosa population pupae. The sample units evaluated were the pot, plant, and leaf. These three units can be used to report the total number of E. formosa pupae in a production batch. A total of 19 pots with three plants each and having 90 leaves were randomly collected from different production batches that were obtained under the conditions described.

The total number of pupae for each sample type was counted, and the data were recorded in a spreadsheet and randomized in a table of two columns; a random function was used to avoid sampling bias. The first column of the table denominated the number of the sample and was organized from one to the maximum value. The second column represented the total number of pupae in each sample. Then, third and fourth columns were calculated based on the accumulative mean and standard deviation, and the sample numbers ranged from sample two to the maximum value. Next, the data calculated for each sample type were plotted using an effort curve as previously described. Finally, the total production of a batch was estimated by multiplying the mean number of pupae from the sample leaves randomly collected from the batch by the total number of leaves that were harvested (Eq. 3).

$$ {N}_T={P}_m\ \mathrm{x}\ {N}_h $$
(3)

where NT is the total number of pupae in a batch, Pm is the number of pupae in a leaf sample, and Nh is the total number of leaves in a batch at the end of production.

Production efficiency of E. formosa

For the purpose of this work, production efficiency was defined as the relationship between the number of new individuals produced and the number of adults released during the mass rearing process (produced/released). This ratio is used to measure the efficiency of mass rearing and indicates how many new individuals are produced for those released. This ratio should not be confused with the net reproduction rate, although they are similar. A value of one for this ratio means that the individuals of E. formosa that are released have exactly enough progeny to replace them and continue the cycle, but no new individuals are produced for sale. A ratio less than one suggests that there are losses of individuals during a certain step of the process. For mass rearing production, values higher than one are needed. This ratio implicitly includes E. formosa fecundity, parasitism rate, and immature mortality. These are important parameters for mass-reared natural enemies (van Lenteren & Tommasini 2003).

To estimate the efficiency ratio, the number of released E. formosa adults was recorded for 17 production batches (one batch = one production cycle) that were reared in the same production scheme as before. The batches were monitored until harvest, at which time the number of pupae produced per leaf was registered. The data were fitted to a second-degree polynomial regression model with the efficiency ratio as the dependent variable and the number of released E. formosa as the explanatory variable (Hothorn & Everitt 2009). The analysis was performed using the lm procedure in R.

Results

T. vaporariorum population density estimation

Estimation of the total number of third instar nymphs per leaf

The mean density of T. vaporariorum third instar nymphs was 27.5 ± 1.94 nymphs/cm2 ranging from 10.8 to 49.6 nymphs/cm2. It is possible to produce as many as 3241 ± 232 nymphs when the mean leaf area is 118 ± 2.47 cm2. The total number of nymphs on the leaf was estimated by multiplying the density (nymphs/cm2) by the total leaf area. The total number of nymphs of T. vaporariorum on the whole leaf (N) is significantly positively related to the density (Nc) and the leaf area available for feeding (A) (Eq. 4) (Fig 1).

$$ N=-3019.16+25.63A+117.4 Nc $$
(4)
Fig 1
figure 1

Surface plot of the total number of third instar nymphs of Trialeurodes vaporariorum in a leaf as a function of the density and leaf area as described in Eq. 4: N =  − 3019.16 + 25.63A + 117.4Nc. R2 = 98.93%, p value < 2.2e−16, n = 30.

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Estimation of the sample size for leaf area and density of nymphs

The minimum number of samples that must be collected in a production batch to estimate the mean leaf area and the population density was calculated using Eqs. 1 and 2. The estimated values were 13.5 samples for the leaf area and 14.5 samples for the density (nymphs/cm2), which is supported by the graphic analysis (vertical lines in Fig 2).

Fig 2
figure 2

Graphical analysis of the minimum sampling size for the parameters leaf area (a) and density (b). X, mean of the parameter in each sample size; XT, mean of the parameter at the maximum sample size. Vertical line, minimum sample size. Error bars, standard deviation.

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Estimation of the minimum sample size and sample unit for E. formosa

The approximate mean number of pupae in each sample unit was 7045 ± 93 for “Pot,” 2350 ± 30 for “Plant,” and 1500 ± 14 for “Leaf.” Graphical analysis showed that the minimum sample sizes were 13 pots (Fig 3a), 42 plants (Fig 3b), and 54 leaves (Fig 3c). Each type of sample unit could be used to determine the number of pupae in a production batch; however, the leaf is a practical sample unit because counting the number of pupae on 54 leaves is an activity that can be performed in parallel with the harvest during the last stage of the production process, where it is necessary to remove and count the total number of leaves per batch. This count requires approximately 1 h per week and can be performed without equipment because pupae are black in color and can be differentiated easily. The counting cost is 1.3 USD per sampling event.

Fig 3
figure 3

Graphical analysis of the minimum sampling size for each sample unit for the mean number of pupae parameter. a Pot. b Plant. c Leaf. X, mean of the parameter in each sample size; XT, mean of the parameter at the maximum sample size. Vertical line, minimum sample size. Error bars, standard deviation.

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Evaluation of production efficiency of E. formosa adults

The relationship between the number of produced E. formosa and the number of released E. formosa was, on average, 3.82. Variation was high among different production process cycles, with values between 1 and 8.9 and a coefficient of variation of 0.58. An inverse relationship was established with higher production efficiency when fewer adults were released (Fig 4).

Fig 4
figure 4

Number of Encarsia formosa produced in function of the density of wasps released (production efficiency ratio) in different production batches. y = 1e−08 x2 – 0.0007x + 12.867 R2 = 0.822.

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Discussion

In this study, a method to scout populations in a tritrophic mass rearing facility was adapted using the leaf area of the plant and the population density of insects as main parameters. Standardizing this kind of method is important because it could help producers accurately determine the required production and detect possible anomalies in the process; additionally, it is possible to estimate certain costs using the data that are collected during scouting.

The density parameter found in this research could be used as an indicator for the production and as a standard for other mass rearing producers, but it is important to clarify that this parameter was measured using common bean plants for the rearing process; thus, parameter values could vary for other host plant species. The value found in this study ranged from 10 to 50 nymphs/cm2 and could be explained by the high number of adults used to infest the leaves and the high supply of nitrogen through fertilization that has a positive effect on oviposition (Jauset et al1998, 2000, Ortega-Arenas et al2006, Park et al2009).

No previous studies have measured the population density of T. vaporariorum nymphs under mass rearing conditions using common bean as the host plant. Some authors have measured the density in commercial crops using other host plant species (i.e., tomato, gerbera, strawberry, and tobacco). Due to this lack of information for mass rearing, we use the data from these commercial crops to analyze the variability of the density parameters as shown below.

Ortega-Arenas et al (2006) reported that T. vaporariorum population density could range from 2.7 to 13.9 nymphs/cm2 in Gerbera jamesonii (Bolus ex Hook.f.) (Asterales: Asteraceae) plants, depending on the plant cultivar and its nitrogen concentration. The whitefly population was larger when the nitrogen fertilization level is high. Bi et al (2002) found that the maximum population density in strawberry plants under cover was 12 nymphs/cm2. In greenhouse tomato crops, values ranging from 4 to 30 nymphs per leaf have been reported for a minimum leaf area of 100 cm2 (Jauset et al2000, Basso et al2001, Park et al2009). In string bean and common bean, Bueno et al (2005) reported that when adults covered 60% of the infested leaf area, the population density of nymphs was 25.2 nymphs/cm2.

Those studies present population density values that vary in the range determined in the present research, and those other values suggest there is a host plant effect on the density of nymphs. No information about the density parameter in tobacco plants was found, although it has been reported as the best plant for mass rearing whiteflies (Scopes & Biggerstaff 1971). Oviposition preference for whiteflies have been reported to correlated positively with leaf morphology characteristics as leaf trichome density, trichome length, and leaf lamina thickness (Hasanuzzaman et al2016), so differences in oviposition between host plants also could be related to leaf morphology.

The monitoring cost in this study was approximately 1.60 USD for the 54 leaves (0.03 USD/leaf), and approximately 1-h labor was required to perform the sampling. This activity must be performed weekly. No previous studies have estimated this cost under mass rearing conditions; however, Bernal et al (2008) estimated that the monitoring cost for T. vaporariorum nymphs in commercial greenhouse tomato crop was 1.00 USD at the density of 12 nymphs per leaf with a precision level of 5%, assuming a sample size of 198 leaves (0.0050 USD/leaf). The monitoring cost per leaf found in this study was higher than that reported under commercial conditions, which was possibly due to the higher density of nymphs on the common bean leaf under mass rearing. Although this cost was higher, it is necessary to complete this task to estimate the population level in the mass rearing.

The efficiency ratio stablished in this work (Fig 4) shows that as the number of E. formosa released in a production cycle increased, the number of E. formosa adults that were produced diminished, suggesting that the optimal number should be approximately 7000 in a weekly production batch. Similar results were reported by Berndt & Meyhöfer (2007), which analyzed the control efficiency of E. formosa released at high densities (1.000) over a T. vaporariorum population in Gerbera. According to the authors, the use of this high E. formosa density caused a decrease in the rate of parasitism, which was possibly related to the parasitoid selection process for oviposition and/or predation (i.e., host-feeding) of the prey. Netting & Hunter (2000) reported that E. formosa females are able to recognize nymphs of T. vaporariorum that have been previously parasitized, and instead of discarding the host, they kill the egg from their congener to oviposit their own eggs. The authors also reported that this behavior has no significant effect on the handling time; thus, the effect of interference found in this study is not due to the loss of search time between parasitized and non-parasitized hosts but rather to the ovicidal behavior among congeners, which will increase if the E. formosa population is very high.

Previous authors have used the term “efficiency” to refer to the parameters of time or cost in natural enemy rearing conditions. Nasreen et al (2011) established that the production efficiency for a Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) rearing system was the proportion between the reproductive potential (total fecundity) and the development time. Using this concept, the authors estimated the production of females per time unit, which they reported was affected positively by prey egg quantity. De Bortoli et al (2011) used efficiency, but they used cost terms rather than time. They established that the efficiency of the process was affected by the net reproduction rate (Ro), and the efficiency was higher when the Ro values were high. In the present research, neither time nor cost was used to establish efficiency; rather, a gross marginal index was used because it helps better understand how productive the rearing in numbers was, i.e., how many insects i produced for each one i used.

In a tritrophic system, population parameters are strongly related between species; for example, it has been reported that the number of E. formosa adults to be released depends on the number of T. vaporariorum third instar nymphs present (Tello et al2007), and a 1 to 17 ratio is preferred. This optimized relationship maximizes the parasitization activity and prevents interference between E. formosa adults (Netting & Hunter 2000). This reported ratio was used in this research and is supposedly to allow a 77.13% rate of parasitism (Aragón et al2008). This same proportion was reported by Roermund & Lenteren (Van Roermund et al1997), who used a simulation model to establish that the maximum parasitic capacity of E. formosa on T. vaporariorum was 17 nymphs. Considering these previous reports, it is possible to use the data in the “T. vaporariorum Population Density Estimation” section to estimate that the number of E. formosa to be released per leaf must be 190 E. formosa (3241 nymphs/17 E. formosa).

Finally, the consolidated sampling method can be resumed in six steps as follows. (1) Take a random sample of 14 leaves from a production batch with third instar nymphs; (2) calculate the density of third instar nymphs on a 1-cm2 section of the leaves and measure the leaf area; (3) use Eq. 4 to calculate the mean number of nymphs on a leaf and multiply this value by the approximate number of leaves in the batch (each plant in the batch has two leaves); (4) calculate the number of E. formosa adults to release, factoring the total number of nymphs in the batch into 17; (5) when it is time to harvest the E. formosa pupae, take 54 leaves of the plant and calculate the mean number of E. formosa pupae on the leaves; and (6) calculate the total production using Eq. 4.

To estimate the total population of T. vaporariorum on a leaf, it is necessary to determine the density of nymphs (nymphs/cm2) and the area of the leaf from the samples in a production batch. The results of this study suggest that the optimal number of E. formosa released in each batch depends on the T. vaporariorum density, which should be approximately 27.5 individuals/cm2 for a leaf with an area of 118 cm2. At this density, the number of E. formosa adults released on a leaf should be approximately 190 individuals. When high populations of E. formosa are released in a batch, the production efficiency decreases, producing fewer new adults per adult used in the batch.