Introduction
Temperature affects the physiological and biochemical processes in ectothermic animals, such as insects and is a determining factor in the developmental time, survival, and distribution of a species (Cossins and Bowler, Reference Cossins and Bowler1987). Changes in temperature induce insect adaptations that trigger metabolic costs (Régnière et al., Reference Régnière, Powell, Bentz and Nealis2012). Moreover, insect responses to changes in temperature have been characterised as a trade-off for energy allocation. Amongst the physiological functions, nutrients are allocated for maintenance, reproduction, and survival (Boggs, Reference Boggs2009). Furthermore, it is expected that gradual temperature changes induce physiological responses to aid organism survival in their habitat (Kingsolver and Huey, Reference Kingsolver and Huey1998) that are associated with a direct fitness cost (Milosavljević et al., Reference Milosavljević, McCalla, Ratkowsky and Hoddle2019). However, natural enemy colonies cultivated in insectaries are carefully managed in optimal environmental conditions, ensuring the efficient mass production of insects within a short timeframe (Chown and Nicolson, Reference Chown and Nicolson2004; Ikemoto, Reference Ikemoto2005; Ortiz et al., Reference Ortiz, Torres Ruiz, Morales-Ramos, Thomas, Rojas, Tomberlin, Yi, Han, Giroud, Jullien, Dossey, Morales-Ramos and Guadalupe Rojas2016). Therefore, gradual change of temperature in rearing facilities before releasing natural enemies could increase their survival under different thermal conditions in the target habitat, and conserve their reproductive potential (Kristensen et al., Reference Kristensen, Hoffmann, Overgaard, Sørensen, Hallas and Loeschcke2008; Angilletta et al., Reference Angilletta, Condon and Youngblood2019).
Lady beetles (Coleoptera: Coccinellidae) represent one of the most diverse groups of natural enemies of insect pests (Dixon, Reference Dixon2001). Research on this group has shown the impact of temperature on fecundity and fertility (Sørensen et al., Reference Sørensen, Toft and Kristensen2013; Zhang et al., Reference Zhang, Cao, Wang, Zhang and Liu2014; Ferreira et al., Reference Ferreira, Silva-Torres, Venette and Torres2020; de Oliveira et al., Reference de Oliveira, Torres, Torres and Silva2022), body size (Sørensen et al., Reference Sørensen, Toft and Kristensen2013; Maes et al., Reference Maes, Grégoire and De Clercq2015), developmental time, survival (Sørensen et al., Reference Sørensen, Toft and Kristensen2013; Maes et al., Reference Maes, Grégoire and De Clercq2015; Sebastião et al., Reference Sebastião, Borges and Soares2015; Ferreira et al., Reference Ferreira, Silva-Torres, Venette and Torres2020), number of generations, and the potential global geographic distribution (Ferreira et al., Reference Ferreira, Silva-Torres, Torres and Venette2021). Research has characterised the potential of the predatory lady beetles to control the target pest in new habitats (Obrycki and Kring, Reference Obrycki and Kring1998; Kim and Lee, Reference Kim and Lee2008). For instance, when lady beetles were found under optimal temperature, which usually varies around 20°C (Dixon et al., Reference Dixon, Honěk, Keil, Kotela, Šizling and Jarošík2009) depending on the species, they showed great fitness (Ferreira et al., Reference Ferreira, Silva-Torres, Venette and Torres2020, Reference Ferreira, Silva-Torres, Torres and Venette2021). When exposed to extreme temperature conditions, they may prioritise a specific physiological function to favour their survival or their offspring (Blanckenhorn, Reference Blanckenhorn, Córdoba-Aguilar, González-Tokman and González-Santoyo2018).
The lady beetle Tenuisvalvae notata (Mulsant) (Coleoptera: Coccinellidae), from South America, has contributed to the management of mealybugs (Hemiptera: Pseudococcidae) in Brazil and other countries (Dreyer et al., Reference Dreyer, Neuenschwander, Bouyjou, Baumgärtner and Dorn1997a, Reference Dreyer, Neuenschwander, Baumgärtner and Dorn1997b; Barbosa et al., Reference Barbosa, Oliveira, Giorgi, Oliveira and Torres2014; Peronti et al., Reference Peronti, Martinelli, Alexandrino, Júnior, Penteado-Dias and Almeida2016). It is distributed in tropical and subtropical regions, with an optimal temperature range of 25–28°C, and maximum and minimum temperature thresholds of 35 and 14°C, respectively (Dreyer et al., Reference Dreyer, Neuenschwander, Baumgärtner and Dorn1997b; Ferreira et al., Reference Ferreira, Silva-Torres, Venette and Torres2020). Despite the wide range between maximum and minimum temperature thresholds, recent studies have shown that T. notata can survive between 20 and 36°C but reproduce only below 32°C (Ferreira et al., Reference Ferreira, Silva-Torres, Venette and Torres2020, Reference Ferreira, Silva-Torres, Torres and Venette2021), possibly due to male sterilisation at high temperatures. Furthermore, when fed on Ferrisia dasylirii (Hemiptera: Pseudococcidae), de Oliveira et al., Reference de Oliveira, Torres, Torres and Silva2022 found that the pre-oviposition period of T. notata raised at 28°C varies from 1.3 ± 0.21 to 1.8 ± 0.27 days. In addition, the oviposition period can vary from 50 to 86.9 ± 7.27 days, depending on the presence of males and females, given the same temperature and prey conditions. This allows for many copulations throughout the adult life (Tuler et al., Reference Túler, Silva-Torres, Torres, Moraes and Rodrigues2018; de Oliveira et al., Reference de Oliveira, Torres, Torres and Silva2022).
Other studies have suggested that a reduction in insect reproduction can be related to effects of temperature variation on spermatogenesis, through direct injuries on testicles, and seminal vesicles being emptied, or with a reduced amount of sperm as observed in Diptera, Coleoptera, and Hymenoptera insects (David et al., Reference David, Araripe, Chakir, Legout, Lemos, Petavy, Rohmer, Joly and Moreteau2005; Nguyen et al., Reference Nguyen, Rieu, Mariani and van Dam2016; Sales et al., Reference Sales, Vasudeva and Gage2021), and such aspects need to be further investigated in lady beetles such as T. notata. In this context, the hypotheses in our study were: (i) If a trade-off occurred in T. notata adults subjected to different temperatures, thus female survival would be favoured at the expense of fecundity and fertility, and (ii) If temperatures out of the optimal range affect T. notata reproduction, then females coupled with males under extreme temperatures would suffer a fertility decline.
Temperatures used here were classified as extreme for the reproduction and survival of this species (Ferreira et al., Reference Ferreira, Silva-Torres, Venette and Torres2020, Reference Ferreira, Silva-Torres, Torres and Venette2021). Results from this research will help us understand the physiological plasticity of T. notata regarding its adaptation and establishment under different temperature conditions and how this could be used as a tool in rearing facilities aiming to improve survival rates of T. notata releases in the field.
Material and methods
Study site
This study was carried out at the Laboratory of Insect Behavior of the Universidade Federal Rural de Pernambuco (UFRPE), Recife, Pernambuco State, Brazil. The mealybug Ferrisia dasylirii (Cockrell) (Hemiptera: Pseudococcidae) was collected from cotton plants in the experimental area of the UFRPE (Recife, Pernambuco, Brazil, 8.017070°S, 34.944362°W), and identified by Vitor Cezar Pacheco da Silva (Universidad de la República, Uruguay). Tenuisvalvae notata was collected from cotton plants infested with the cotton mealybug Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) (Surubim, Pernambuco, Brazil, 7.8330°S, 35.7547°W), and identified by José Adriano Giorgi (Universidade Federal do Pará, in memoriam). The colonies were maintained under laboratory conditions (28 ± 2°C, 65 ± 5% R.H., 12 L: 12D), previously described as ideal for reproduction and maintenance of those insect species (Ferreira et al., Reference Ferreira, Silva-Torres, Venette and Torres2020).
Rearing of prey and predator
The mealybug (F. dasylirii) colony was reared on pumpkins (var. Jacarezinho), following protocols by Sanches and Carvalho (Reference Sanches and Carvalho2010), and adapted to our conditions (Oliveira et al., Reference Oliveira, Silva-Torres, Torres and Oliveira2014). Briefly, the pumpkins were infested by gravid females and the mealybug colony was allowed 30 days to establish and to be fully covered with the mealybug colony. Mealybug-infested pumpkins were offered to T. notata in transparent Plexiglass cages (40 × 25 × 20 cm), where the lady beetle completed its development. Pupae of T. notata were collected and placed in plastic Petri dishes (5 cm diam.). Emerged adults were separated by sex morphological differences (females have two black spots on the head between the eyes and are usually larger than males (Barbosa et al., Reference Barbosa, Oliveira, Giorgi, Oliveira and Torres2014)) and kept individually in same size Petri dishes (5 cm diam) before submitting them to gradual change of temperature treatments.
Survival and reproduction under gradual change of temperature conditions
This experiment aimed to evaluate the reproduction and survival of T. notata adults submitted to gradual thermal changes of high and low temperatures. The temperatures were chosen based on the previous studies by Ferreira et al. (Reference Ferreira, Silva-Torres, Venette and Torres2020) and Dreyer et al. (Reference Dreyer, Neuenschwander, Bouyjou, Baumgärtner and Dorn1997a) that showed a limit of development and reproduction of T. notata under temperatures below 18°C and above 36°C.
Thus, adults (<24 h old) were subjected to gradual change of temperature in growth chambers (BOD (biochemical oxygen demand) incubator), by increasing or reducing the temperature at the rate of 1°C per day, which reached the maximum/minimum temperatures treatment as follows. Low-temperature treatments were 12, 14, 16, and 18°C, whereas high-temperature treatments were 32, 34, 35, and 36°C, respectively. The optimum temperature treatment was 28°C (Ferreira et al., Reference Ferreira, Silva-Torres, Venette and Torres2020). When the temperature of each treatment (low or high) was reached, couples (> 20) were formed and allowed to mate for 24 h. Therefore, the age of adults at mating depended on which temperature treatment they were subjected to, for instance, adults of optimum temperatures were 24 h old, whereas adults subjected to 36°C were 7 days old. This age difference was not expected to affect results as Túler et al. (Reference Túler, Silva-Torres, Torres, Moraes and Rodrigues2018) showed that mating age up to 100 days is not a significant factor in reproduction for T. notata, as ageing up on this limit did not affect mating behaviour, fecundity, or fertility in this species, as long as the male stays with the females and they copulate multiple times. In our experimental setting, females were separated from males after mating and kept under the same temperature condition after gradual change of temperature. The daily number of eggs, egg hatching, survival, and time of oviposition was recorded for 60 days (according to Túler et al., Reference Túler, Silva-Torres, Torres, Moraes and Rodrigues2018) from n = 20 females that had oviposition. Females that did not oviposit for the first 10 days (peak oviposition time according to Tuler et al., Reference Túler, Silva-Torres, Torres, Moraes and Rodrigues2018) were not considered in the analyses, as they might not have copulated with paired males during the 24 h mating period allowed in the BOD.
Impact of low and high temperature on fertility
Virgin adults of T. notata (male and female) 5–10 days old were subjected to gradual change of temperatures of 16, 18, 32, and 34°C, respectively, starting from the optimum temperature treatment of 28°C. In order to understand whether high and low temperatures would cause male or female sterilisation or reduce reproduction at some extent, couples were formed according to the following treatments: (i) To measure temperature effect on males: females originated from 28°C rearing condition vs males from low (16°C (n = 16), and 18°C (n = 16)) and high temperatures (32°C (n = 12), and 34°C (n = 13)) (fig. 1a), (ii) To measure temperature effect on females: males from 28°C rearing condition vs females from low (16°C (n = 20), and 18°C (n = 13)) and high temperatures (32°C (n = 12), and 34°C (n = 18)) (fig. 1b). The established pairs of male and female mated at 28°C under laboratory conditions and prey abundance. To make sure adults mated, this time couples were observed until the first mating and after that males were removed. The females were placed under their gradual change of temperature or rearing temperature conditions in the BOD and fecundity, egg hatching rate, and time of oviposition were recorded for 60 days.
Figure 1. Scheme of crossings between male and female Tenuisvalvae notata to access the effects of high and low temperatures on males (A) and females (B) reproduction.
Fertility under low temperatures
This experiment evaluated the effects of low temperature and male presence or removal on fertility (measured as egg hatching). Thus, after adult emergence, T. notata were subjected to gradual changes of temperature down to 16°C, as previously described. Pairs were formed and allowed to mate in a Petri dish (5 cm diameter) and kept under the corresponding rearing temperatures. These males and females were assigned to the following treatments: (i) mating pairs were separated after 24 h (male removal), (ii) mating pairs were maintained together for 20 days (male presence). Each treatment had 20 replicates. Female oviposition was tallied daily, 50% of eggs were subjected to the optimal temperature of 28°C (control) and 50% of eggs subjected to the low temperature of 16°C. Egg hatching was recorded daily according to the female condition (alone or paired) as well as temperature (control or low temperature) to ascertain fertility.
Statistical analysis
Data on female survival and time of oviposition were subjected to the Weibull frequency distribution through the ‘survival’ package in R software (R Development Core Team, 2020). Differences between treatments were evaluated by aggregating factor levels until the consequential change in deviance was significant at (P < 0.05).
Data on female fecundity and egg hatching rate were subjected to deviance analysis (ANODEV) through the generalised linear model (GLM) with Poisson distribution, with dispersion corrected by the quasi-Poisson function, followed by an analysis of residuals to verify the adequacy of error distribution and model construction. The data of egg hatching was subjected to ANODEV through GLM with the binomial distribution. The difference among treatments was evaluated through the analysis of contrast (P < 0.05), as described by Crawley (Reference Crawley2007) in R software (R Development Core Team, 2020).
Results
Reproduction and survival under gradual change of temperature conditions
Lady beetles exposed to gradual change of temperature at either high (above 28°C) or low (below 20°C) temperatures exhibited a significant reduction in fecundity. There was a reduction in the number of eggs laid when females were exposed to temperatures below (F 4, 95 = 179.93, P < 0.001; fig. 2a) and above 28°C (F 4, 95 = 65.49, P < 0.001; fig. 2c). Moreover, egg hatching rate was also affected by higher (χ2 = 40.87, df = 4, 95, P < 0.001; fig. 2b) and lower temperatures (χ2 = 51.45, df = 4, 95, P < 0.001; fig. 2c). When lady beetles were subjected to higher temperatures, egg hatching was significantly reduced with few eggs hatched at 35°C and no eggs hatched at 36°C. Similarly, when lady beetles were subjected to lower temperatures was a decrease on hatched eggs at 18°C, and no egg hatching was observed at temperatures lower than 18°C. Female fecundity (F 8, 170 = 86.85, P < 0.001) and egg viability (χ2 = 88.47, df = 8, 170, P < 0.001) were significantly different when we compared all temperature treatments (table 1).
Figure 2. Average (± SE) number of eggs laid (fecundity) by Tenuisvalvae notata and egg hatching (fertility) after gradual change to high (a, b) and low (c, d) temperatures during 60 days observation period.
Table 1. Reproduction traits of Tenuisvalvae notata fed Ferrisia dasyrilii and exposed to different temperatures (°C) for 60 days
* Means followed by the same lowercase letter in the column are not significantly different (α = 0.05).
The oviposition period was prolonged when females were exposed to 18°C (57.3 ± 0.9 days) and 16°C (52.6 ± 1.4 days). A sharp reduction in oviposition period was observed, on average, of 13.8 ± 1.1, and 11.4 ± 1.4 days, when females were subjected to 14 and 12°C, respectively (χ2 = 144.28, df = 4, 95, P < 0.001; fig. 3, left arrow). In contrast, the oviposition period was significantly reduced with the increase in temperature from 28 to 36°C (χ2 = 155.36, df = 4, 95, P < 0.001; fig. 3, right arrow).
Figure 3. Average (±SE) oviposition days of Tenuisvalvae notata, under gradual change of temperature either at low temperatures or at high temperatures, during 60 days observation period. The dashed bar indicates the optimal temperature.
Lady beetle survival was not affected by gradual change in temperature at low temperatures as females lived equal time spans for the whole evaluation period of 60 days (χ2 = 4.3, df = 4, 95, P = 0.37). In contrast, survival was affected when the lady beetles were subjected to high temperatures: survival decreased significantly to less than 30 days at 35 and 36°C (χ2 = 132.06, df = 4, 95, P < 0.001; fig. 4). The temperatures 12 and 14°C showed significant difference compared to 32°C (χ2 = 296.91, df = 8, 170, P < 0.001), whereas those temperatures did not differ from 35°C.
Figure 4. Survival (%) of Tenuisvalvae notata females on different gradual change of temperature during 60 days observation period (censored Weibull model). Shape parameter is taken as α = 2.29.
Impact of low and high temperature on fertility
Regardless of whether females were subjected to low (F 2, 50 = 128.4, P < 0.001) or high (F 2, 47 = 29.56, P < 0.001) temperatures, they laid fewer eggs compared to females kept at 28°C (fig. 5a). Similarly, when only males were subjected to gradual change of temperature, there was a significant reduction in female fecundity at high (F 2, 49 = 65.51, P < 0.001) and at low temperatures (F 2, 42 = 21.625, P < 0.001; fig. 5b). Also, fecundity at 18°C (50.61 ± 7.19) and 34°C (71.83 ± 7.89) was significant different from 16°C (44.37 ± 3.91), (F 4, 98 = 84.615, P < 0.001).
Figure 5. Average (±SE) fecundity and fertility of Tenuisvalvae notata exposed to low (16°C and 18°C, grey bars) and to high (32 and 34°C, empty bars), after gradual change of temperature and paired to a partner (male or female) from the control temperature 28°C (dashed bars).
Note: In a, females were in temperature treatments, whereas in b males were exposed to temperature treatments before mating.
Regarding the egg hatching, there was a significant reduction when females were subjected to gradual thermal changes at low temperatures, and eggs did not hatch at 16°C (χ2 = 32.52, df = 2, 50, P < 0.001; fig. 5a). In contrast, there was no significant effect on fertility when only males were exposed to low temperatures (χ2 = 1.18, df = 2, 42, P = 0.556; fig. 5b). Similarly, there was no effect of high temperatures on fertility for females (χ2 = 0.43, df = 2, 47, P = 0.805) and males (χ2 = 2.13, df = 2, 49, P = 0.345). Also, egg viability was significantly different (lower) at 18°C (102.46 ± 9.10) and 16°C (79.75 ± 8.52) compared to 32°C (44.37 ± 3.91) and 34°C (46.68 ± 8.37), (F 4, 72 = 36.181, P < 0.001).
Overall, there was a significant reduction in the average number of oviposition days when female and males were subjected to low and high temperatures, in comparison to females maintained under 28°C (fig. 6). Whereas females reared at 28°C oviposited for 49.3 ± 3.4 days, females from low temperatures oviposited for less days, 41.8 ± 4.6 days at 18°C, and for 42.0 ± 1.9 days at 16°C (χ2 = 7.23, df = 2,48, P = 0.027). Similarly, females from high temperatures oviposited for 45.2 ± 3.3 days at 32°C and for 38.0 ± 2.0 days at 34°C differing from females reared under optimal temperature of 28°C (χ2 = 9.66, df = 2, 47, P < 0.0001; fig. 6a). In addition, females paired to males from 16°C (subjected to temperature treatment) reduced the oviposition time to 38.6 ± 3.9 days (χ2 = 7.28, df = 2, 42, P = 0.026), for 23.3 ± 2.8 days, and 16.4 ± 2.5 days when mated with males at 32°C and 34°C, respectively (χ2 = 45.86, df = 2, 49, P < 0.0001; fig. 6b). Finally, the oviposition period at 16°C showed a significant difference compared to 32 and 34°C (χ2 = 11.25, df = 4, 76, P = 0.024).
Figure 6. Average (± SE) of reproductive days of Tenuisvalvae notata exposed to low (16°C and 18°C, grey bars), and to high (32 and 34°C, empty bars), gradual change of temperature and paired to a partner (male or female) from the control temperature 28°C (dashed bars).
Note: In A, females were in temperature treatments, whereas in B males were exposed to temperature treatments before mating.
Sperm supply on female fertility under low temperatures
Temperature at which eggs were subjected after oviposition significantly affected egg hatching rate (χ2 = 73.98, df = 1, 137, P < 0.001), as well as surrounding temperature of the adult after emergence (χ2 = 8.035, df = 1, 136, P = 0.0045). However, there was no effect of the presence of males on female fertility (χ2 = 0.196, df = 1, 135, P = 0.66), nor the interaction of these factors (χ2 = 0.238, df = 1, 134, P = 0.63).
Finally, when both parents were subjected to the optimal temperature of 28°C and the eggs were at the same temperature, egg hatching was about 80%. In contrast, when those eggs were incubated at 16°C, there was a significant reduction in egg viability to almost no egg hatching (fig. 7). Moreover, when parents were subjected to 16°C and eggs placed at 28°C, egg hatching was also significantly reduced in comparison to those kept under optimal conditions, and eggs did not hatch at 16°C.
Figure 7. Percentage of egg hatching (±SE) when subjected to two different temperatures (28° or 16°C) of parental development. Means are significantly different (P < 0.001).
Discussion
Abiotic factors, in particular temperature variation, can alter behavioural and physiological parameters of insects, with influence in the population growth of insects (David et al., Reference David, Araripe, Chakir, Legout, Lemos, Petavy, Rohmer, Joly and Moreteau2005). Additionally, insect behaviour, development, and reproduction are favoured within an optimal temperature range between 15 and 30°C, been species-specific and shaped by the exact range of experimental temperatures (Chown and Nicolson, Reference Chown and Nicolson2004). The survival of natural enemies, such as many species of lady beetles is negatively affected above 32°C (Jalali et al., Reference Jalali, Tirry and De Clercq2009, Reference Jalali, Sakaki, Ziaaddini and Daane2018; Yang et al., Reference Yang, Liu, Wyckhuys, Yang and Lu2022). However, in the present study, T. notata adults survived in a temperature range of 12 to 36°C, suggesting that the thermotolerance of this species is stronger, and supporting Ferreira et al. (Reference Ferreira, Silva-Torres, Venette and Torres2020), as 36°C is the maximum threshold temperature for T. notata survival. On the other hand, T. notata has been reported in places of high temperatures in the semiarid region of Pernambuco – Brazil (Barbosa et al., Reference Barbosa, Oliveira, Giorgi, Oliveira and Torres2014; Torres and Giorgi, Reference Torres and Giorgi2018), which suggests that the species can present behavioural and physiological strategies to survive warmer weather conditions up to a limit of 36°C. Nevertheless, both fecundity and fertility of T. notata were affected by low and high temperatures studied. Individuals that spend more energy on reproduction usually have a reduction in longevity in comparison to those that do not mate and reproduce (Omkar and Mishra, Reference Omkar and Mishra2005; Dixon et al., Reference Dixon, Honěk, Keil, Kotela, Šizling and Jarošík2009; Mirhosseini et al., Reference Mirhosseini, Michaud, Jalali and Ziaaddini2014), and this is known as a survival × reproduction trade-off. Thus, it is expected that after the gradual temperature change T. notata can survive, but have a negative impact on fitness, in places where the temperature is outside its optimal range of 20–32°C (Ferreira et al., Reference Ferreira, Silva-Torres, Venette and Torres2020). In this context, adults of T. notata were able to survive in temperatures up to 36°C, and females were able to lay eggs for about 10 days, but without egg hatching.
Colder environmental conditions were associated with an extended duration of copulation and higher transmission rates of sperm (Kristensen et al., Reference Kristensen, Hoffmann, Overgaard, Sørensen, Hallas and Loeschcke2008; Wang et al., Reference Wang, Tan, Guo and Zhang2013; Maes et al., Reference Maes, Grégoire and De Clercq2015). The current study shows that at low temperatures (<18°C), T. notata females subjected to a gradual reduction in temperature after emergence were able to survive and reproduce but had a reduction in the total oviposition period, fecundity, and fertility, as was observed in another lady beetle species (Wang et al., Reference Wang, Tan, Guo and Zhang2013). Some lady beetle species from temperate regions enter diapause or quiescence as adults, induced by a reduction in the photoperiod (Sakaki et al., Reference Sakaki, Jalali, Kamali and Nedvěd2019; Obrycki, Reference Obrycki2020), in response to the limitation in food availability and quality as a function of environmental conditions encountered by the insects (Michaud and Qureshi, Reference Michaud and Qureshi2013). As reported, in Paraguay T. notata was found in areas of freezing (0°C) temperatures and short days during the winter (Dreyer et al., Reference Dreyer, Neuenschwander, Baumgärtner and Dorn1997b) and T. notata preys, such as Maconellicoccus hirsutus and P. solenopsis, develop at temperatures between 15 and 35°C (Barbosa et al., Reference Barbosa, Oliveira, Giorgi, Oliveira and Torres2014; Peronti et al., Reference Peronti, Martinelli, Alexandrino, Júnior, Penteado-Dias and Almeida2016), and benefit from the increase in temperature, with a shorter life cycle and increased reproductive rate, like other species of Pseudococcidae (Oliveira et al., Reference Oliveira, Silva-Torres, Torres and Oliveira2014; Bertin et al., Reference Bertin, Lerin, Botton and Parra2019). Further studies are needed to clarify whether or not T. notata is capable of entering reproductive diapause, or if it has other adaptations that allow it to sustain the population in an area with lower temperatures for longer periods.
In this study, we hypothesised that females that mate with males exposed to high and low temperatures would have a reduction in offspring due to the males' reduced capacity. In contrast, we found that when both sexes were subjected to the gradual change of temperature this reduced fecundity and fertility of T. notata. Especially when T. notata females were exposed to temperatures below 18°C, they had a significant reduction in fitness, even when eggs laid were subjected to the temperature of 28°C after oviposition. These findings suggest that male and female T. notata need favourable temperatures for their development, oviposition, and F1 embryo development (Sales et al., Reference Sales, Vasudeva and Gage2021). In this context, some studies have found that high temperatures may cause thermal shock and injuries on oocytes and ovarian development of insects (Krebs and Loeschcke, Reference Krebs and Loeschcke1994; Rinehart et al., Reference Rinehart, Yocum and Denlinger2000; Zhang et al., Reference Zhang, Cao, Wang, Zhang and Liu2014). However, the effects of thermal stress on insect reproduction cannot be analysed separately by gender, when many studies have shown that each insect species can express its vulnerability to thermal stress differently in males and females (Janowitz and Fischer, Reference Janowitz and Fischer2011; Porcelli et al., Reference Porcelli, Gaston, Butlin and Snook2017). Therefore, studies regarding the direct observation and dissection of T. notata gonads (testicles and ovaries) after the gradual change of temperature are underway to understand the effect of temperature fluctuation on its reproduction. In this regard, there are histological and biochemical alterations in gonads of T. notata F2 adults subjected to low (18°C) and high (32°C) temperatures in comparison to those reared at optimal conditions (28°C) (unpublished data). In addition, when directly exposed to heat sources, an organism is subject to dehydration. In particular, the eggs can't move, but provide structure and a water source for protection against desiccation (Jacobs et al., Reference Jacobs, Rezende, Lamers and van der Zee2013), but depending on the time and intensity of exposure, this structure may not be enough to avoid embryo death. Therefore, if harsh conditions persist for a long time a decline in egg fertility can be expected.
Previous studies have shown that lady beetle eggs are more sensitive to changes in temperature, whereas adults and larvae have behavioural responses (i.e. move to areas of microclimate more favourably) (Chown and Nicolson, Reference Chown and Nicolson2004; Chen et al., Reference Chen, Gols, Biere and Harvey2019). Because T. notata eggs were not viable under temperatures below 16°C, it is expected that over time this would cause a decline in population numbers of this species if temperature conditions do not increase above 20°C. In addition, T. notata subjected to low (18–20°C) temperatures in the laboratory for two consecutive generations and adults released in a greenhouse with temperatures around optimal conditions had impaired reproduction for 30 days (Silva-Torres et al., unpublished data). This is currently under investigation to answer what is causing the reduction in fecundity and the real effects of the temperature in the lady beetles' eggs. Therefore, it is possible that the lower temperatures in this study did not supply enough thermal energy, or nutrients, for embryo development and egg hatch.
In summary, in this study we found that a gradual increase or decrease in temperature at the adult stage affected most of the measured traits and helped to maintain insect adults alive in rearing facilities according to demand for field releases. When T. notata remain for a long period under exposure to temperatures outside the ideal range they present different reproductive responses for each sex to high and low temperatures. Male lady beetles were more affected by high temperatures while the fertility of females was found to be reduced when they are kept at temperatures lower than 20°C. In addition, current studies on the physiology and morphology of the reproductive systems of T. notata will explain how their progeny are being harmed by changes in temperature, not only in the adult phase but also prior to emergence. Further biological and behavioural studies under more natural settings with temperature fluctuation throughout the day and seasons would be interesting to further understand different fitness effects as those found here. This is the first attempt to report a possible temperature acclimation in T. notata adults, and how this could affect its fitness and survival. Therefore, insect's gradual change of temperature to new temperature conditions will promote an adaptive physiological response to favour the insect survival in such places, and this adaptation will be reflected in its population density.
Acknowledgements
We thank Dr Robert W. Matthews (emeritus professor at the University of Georgia – UGA) and William Samson (Graduate Student at the University of California, Riverside) who provided valid insights and English editing of the manuscript, respectively.
Author contributions
EBSC, CSAST, and JBT conceived the idea and conceptualisation, wrote, reviewed, and edited the manuscript. EBSC collected and analysed the data. CSAST conceived supervision and funding acquisition. All authors read and approved the manuscript.
Competing interest
The authors declare none.
