Biological material and D. opuntiae rearing

The O. ficus-indica cladodes of 24 months of age, without symptoms of diseases, were obtained from the nopalitos producing zone in Tlalnepantla, state of Morelos, Mexico, and they were left to heal for 15 d to prevent the development of pathogens. The cladodes were planted in pots of 20 cm diameter with a mixture of tezontle sand and peat moss (Premier^®, Quebec, Canada) in a proportion of 2:1.

The rearing of D. opuntiae was established with specimens collected from the same region (Tlalnepantla) and placed in Petri dishes, which were transported to Colegio de Postgraduados in Montecillo, Estado de Mexico. The gravid females were identified by the presence of a yellow-amber drop on the dorsal section of the abdomen (^{Marín and Cisneros, 1977}). The infestation and rearing method was performed according to ^{Portillo and Vigueras (2008)}: 12 gravid females were placed inside paper envelopes (5(5 cm) that stayed attached to the cladode for two weeks. This material was kept under greenhouse conditions (23±7 °C and 60±20 % relative humidity). Monitoring the life cycle and fecundity experiment was done in a growth chamber (25±1 °C and 40±10 % relative humidity).

Biology and life cycle

Incubation was evaluated in five groups of 20 eggs (each group was considered one repetition) in a completely random experimental design. Each group of 20 eggs, less than 30 min of age, was placed in a Petri dish of 9 cm diameter with moist cotton at the bottom, and on top of it a metallic mesh and dry filter paper were placed. The eggs were placed on the dry filter paper and observations were made every 15 min until hatching (^{Mathenge et al., 2009}).

In order to determine the life cycle, five cladodes were placed inside a growth chamber; each cladode was uprooted one day before beginning the experiment and 10 gravid females were placed on each one. The females were kept there 12 h to obtain nymphs I, then the females were removed and only nymphs I were kept. The cladodes were observed every 12 h until 30 nymphs I per cladode were attached and the remaining nymphs were eliminated. The site of establishment on the cladode of each nymph was marked with indelible ink and the observations for the rest of the cycle were carried out every 24 h. The change in instar was determined by the presence of molt in nymph I and nymph II in females, molt in nymph I and pupa development in males. In addition, the periods of pre-oviposition, oviposition, and sex ratio were recorded. To estimate the development time, 150 individuals (30 per cladode) were evaluated and each individual was considered a repetition, but only data from the individuals that reached the adult state were included.

To describe the basic biology of D. opuntiae on O. ficus-indica, qualitative observations of shape, color, and wax production were performed on 50 individuals, 2 min per individual, in each development stage (eggs, nymphs I, nymphs II, adult females in sexual maturity, pupas and adult males).

Fecundity and longevity

The pre-oviposition of D. opuntiae was measured with 80 females established on five cladodes, 16 females per cladode, detached from the plant and confined to a growth chamber. To ensure mating, two males per female were placed on the cladodes. The end of the pre-oviposition was determined by observing each female daily, lifting it slightly, in search for eggs or nymphs.

The fecundity was evaluated using two methodologies: access to food keeping the females attached to the cladodes (n=40) (^{Moran and Cobby, 1979}; ^{Sullivan, 1990}), or without food by removing the females from the cladodes and placing them individually in Petri dishes (n=40) (^{Flores-Hernández et al., 2006}; ^{Mathenge et al., 2009}). The females attached to the cladode were isolated individually with a removable device made of “fomi” (thermoplastic polymer made up of repetitive units of ethylene and vinyl acetate) and organza fabric (light white cotton fabric) to prevent the escape of the nymphs and favor ventilation. Both groups were checked every 24 h to count nymphs and eggs by removing them during each observation. To record female survival, these remained under observation since the time of removing from the cladodes (without food) or when they were left on them (with food) until the end of the oviposition period. For males, their longevity on the cladodes was recorded. The fecundity and survival were evaluated per treatment in individual females, 40 females for each treatment, that is 40 repetitions, distributed randomly on the cladodes and in the Petri dishes. Using data about D. opuntiae survival, as well as fecundity and sex ratio, a life and fertility table was built to obtain the population parameters.

Data analysis

The average values and standard deviations were calculated for the duration of each stage of development (egg, nymph, pupa and total), for the fecundity and for the survival. The development time of females and males was compared for each instar through the Student t-test (^{SAS Institute, 2000}). The same statistical test was used to compare the fecundity between females with or without food. The survival of females per treatment was compared with the Logrank test (^{Vera et al., 2002}). The fecundity values of specific age per female were used to elaborate a life and fertility table.

The parameters of the life and fertility table of D. opuntiae were estimated through the ^{Birch method (1948)}. The net reproduction rate (Ro), the generation time (T), the intrinsic rate of natural increase (rm) and the finite rate of increase (l) were obtained through the program by ^{Maia et al. (2000)} in the Lifetable SAS software (^{SAS Institute, 2000}), which includes the Jackknife test to estimate confidence intervals for all the parameters. It also performs Student t-tests for the paired groups.

Biology and life cycle

The eggs presented an oval shape and bright red color characteristic of species of the Dactylopiidae family (^{Moran and Cobby, 1979}; ^{Mathenge et al., 2009}). Oviposition can be individual or in group, linked in a chain shape. It is common for the eggs to alternate in different stages of development and even with completely developed nymphs I; 78 % of the eggs hatched and their incubation period was 61.78 ± 24 min, representing less than 3 % of the total cycle. The variation in incubation period is attributed in part to the methodology, which is similar for the species of Dactylopius, where it is not possible to determine the degree of development of the embryos when the eggs are collected because the Dactylopiidae insects present ovoviviparity (^{Marín and Cisneros, 1977}; ^{Gilreath and Smith, 1987}; ^{Mathenge et al., 2009}). The incubation period of D. coccus is 15 to 20 min (^{Marín and Cisneros, 1977}), which represents one third of what was observed in this study; in contrast, ^{Mathenge et al. (2009)} report an incubation period of 17 d in D. tomentosus. In the cases of D. opuntiae and D. coccus, it is likely that the incubation times are underestimated; however, until now new methodologies have not been developed to estimate the real incubation time in other species of Dactylopiidae. This action must be carried out to establish with greater precision this period.

The nymphs I began to produce wax immediately after attaching to the cladode and it was not possible to differentiate the sex through morphological characteristics until after the second instar. The first instar nymph lasted 8.59±1.54 d in males, which was longer than in females (t=2.68, p≤0.01), although the difference was less than 24 h (Table 1). This duration contrasts with reports for the same instar in D. coccus (20-23 d) (^{Marín and Cisneros, 1977}) and D. tomentosus (18 d) (^{Mathenge et al., 2009}). The second instar presented a longer duration in females than in males (t=5.32, p≤0.0001), with a difference of 1.5 d (Table 1). Dactylopius ceylonicus has a similar development period for the same instar (7 d at 26 °C) according to ^{Sullivan (1990)}, but other species such as D. coccus require up to 18 d for this same development stage (^{Marín and Cisneros, 1977}).

After the second instar, only the males can retract the mouthparts, have mobility and seek for a site to develop the pupa; in contrast, the females remain motionless throughout her life with the mouthparts inserted in the cladode. The development time of the pupa was less than 10 d (Table 1). Therefore, the female cycle was shorter than the males’ (t=15.12, p≤0.0001) (Table 1). The duration of the development time in both sexes differs around 15 d for females, compared to the duration reported by ^{Flores-Hernández et al. (2006)} for the same species.

It is likely that the shorter development time in this study is due to the host (O. ficus-indica) and the constant temperature (25±1 °C), since the study by ^{Flores-Hernández et al. (2006)} was done using O. megacantha and greater variations in temperature (19-23 °C); nevertheless, other factors such as the nutritional conditions of the species on which they develop are not dismissed. A higher concentration of calcium oxalate crystals than potassium makes difficult for nymphs to insert their stylets and establish themselves on the cladodes (^{Tovar and Pando, 2010}). Another factor that was not taken into account in this study was the population of D. opuntiae, because the insect populations in each locality could be adapted to the species and variety of nopal on which the studies were carried out independently. ^{Volchansky (1999)} indicated that these differences in population cause variation in the development time.

In the adult stage, the females produce wax with cotton aspect that increases in volume until covering them completely. The adult females matured sexually, they mated and increased the volume of their body, probably because of accumulation of eggs in the ovarioles. The aspect of the adult females is similar to the one described by ^{Sullivan (1990)}, ^{Flores-Hernández et al. (2006)} and ^{Mathenge et al. (2009)}. During the pre-oviposition period, 17.49±2.73 d, a drop of a substance was evident, on the dorsal part, which appears crystalline and then changes to light yellow and amber. This substance is similar to that described in D. coccus (^{Marín and Cisneros, 1977}) and could have a sexual attractant in this species (^{Rodríguez et al., 2005}), but there is no information about this characteristic in other species of the Dactylopiidae family.

In this study, the sex ratio was 3.7:1 (females: males) and was different from the 1:1 ratio reported by ^{Flores-Hernández et al. (2006)} for the same species. According to ^{Nur et al. (1987)}, the sex of scale insects of Pseudococcidae family can be modified by physiological factors of the egg and by environmental conditions. The authors of this study have observed that in the field, the sex ratio of this species is different in specific seasons of the year, although there is no formal record of these changes.

Fecundity and survival in females

The fecundity in females of D. opuntiae (eggs and nymphs) had a response related to the food and the age. The females with food were more fertile (567.58±164.67) than without food (351.25±131.98) (t=6.48, p≤0.005). Additionally, most females began to lay eggs or nymphs after the second week as adults and the oviposition lasted six or nine weeks depending on the availability of food (Figure 1).

Figure 1 Weekly fecundity of D. opuntiae (mean±standard error) with or without food (attached or removed from the O. ficusindica cladodes) at 25±1 °C and 40±10 % of relative humidity 

Both groups of females produced a higher number of eggs and nymphs in the fourth week, during which 52 % of the total fecundity in females without food was accumulated, and 39.92 % in females with food. The females without food laid the highest number of eggs and nymphs from week two to week six (98 % accumulated). The females with food needed two more weeks to lay 98 % of the total eggs and nymphs (Figure 1).

The fecundity differs from what ^{Flores-Hernández et al. (2006)} reported for the same species, who mention an average of 131 individuals per female without food (removed from the cladodes). This difference is attributed to a different host before removing the females from the cladode, although there are situations such as quality, nutrition and stress of the cladodes before withdrawing the females. Two other species of Dactylopiidae evaluated with females attached to the cladodes had higher fecundity; in D. austrinus the progeny was 1,145 individuals female-¹ (^{Moran and Cobby, 1979}) and in D. ceylonicus 1,200 individuals female^-1 (^{Sullivan, 1990}). The reproductive capacity between species of Dactylopius that developed on the Opuntia genus, and the possible differences between the insect-host relationships require further study to understand how the reproductive potential in each species is manifested.

The females with food had a higher survival rate (53.97±5.19 d) compared to the females without food (36.55±2.78 d) (X² =41.29, p≤0.001). This difference in survival of each group of females can be related to the availability of food, but probably also to the physiological demand that the fecundity represented.

Life and fertility table

The survival from egg to adult of D. opuntiae was 64.7 %, of which 17 % of the mortality took place in the egg stage and 17.5 % in nymph I; the population parameters of the species were established with these values plus fecundity and sex ratio (Table 2). The reproductive potential of D. opuntiae on O. ficus indica was higher than the one reported on O. megacantha (^{Flores-Hernández et al., 2006}), but the experiments were developed under different conditions.

With the population parameters, the reproductive potential of D. opuntiae in its principal host in the world was established. The females with food had a net reproductive rate (females’ progeny) and a generation time bigger than the females without food (p≤0.0001), but this did not make their intrinsic rate of increase different (Table 2). This apparent contradiction in the reproductive potential can be explained because the females without food laid the totality of the eggs and nymphs in a shorter time. That is, the availability of food favored the net reproductive rate but increased the generation time (Table 2). These values will be a reference to compare them with its natural enemies, Hyperaspis trifurcata Schaeffer (Coleoptera: Coccinellidae) and Leucopis bellula Williston (Diptera: Chamaemyiidae). However, in population ecology studies, the reproductive potential of the predators will have other parameters that may influence the total response of the natural enemy; for example, the predatory capacity, voracity and searching ability should be added in order to estimate their potential as biological control agents of D. opuntiae (^{Vanegas-Rico et al., 2016}).