Biology and life cycle Of Diatraea busckella ( Lepidoptera: Crambidae ) under simulated altitudinal pro ﬁ le in controlled conditions

: Diatraea busckella ( Lepidoptera: Crambidae ) , a sugarcane borer, was recently reported in Valle del Cauca, Caldas, and Risaralda in Colombia. This fact puts sugarcane ( S. o ﬃ cinarum ) producers in a critical situation. The insu ﬃ cient knowledge about Diatraea spe - cies phenomenology, biology, and the life cycle feature under di ﬀ erent climate scenarios does not let the experts relate ( with certainty ) the population explosion with cli - mate o ﬀ er changes. The work was carried out at the Center for Research and Breeding of Natural Enemies, located in the botanical garden of the University of Caldas at 5.0563885°3 ′ 23 ″ north latitude and − 75.49416675°29 ′ 39 ″ west longitude, at an altitude of 2,160 m above the sea level and an average temperature of 14°C ( Manizales, Caldas ) . To cope with the lack of bio - meteorological infor - mation, an experimental approach, based on the simula - tion of sugar cane production environments, was designed for obtaining the relationship between air temperature and the life cycle of the sugarcane borer. Growth chambers were used to simulate the air temperature of ﬁ ve ambients ( 10, 20, 24.7, 30, and 35°C ) . Under low latitude conditions ( 5°N ) , the referred temperatures corresponded to x , y , z , a , and b . The cycles were completed up to the ﬁ rst oviposi - tion by females; in addition, the direct relationship between air temperature and the insect ’ s development, reproduction, and survival was veri ﬁ ed. The number of days of the larval stage was 40 days DS ± 16.7 ± 2 days from larva to pupae and 6 ± 1 more to reach the adult stage under controlled conditions under ﬁ ve di ﬀ erent tempera - tures ( 10, 20, 24.7, 30, and 35°C ) . In addition, signi ﬁ cant di ﬀ erences were found in development time under con - trolled conditions ( 20, 24.7, and 30°C ) . Diatraea busckella can become a signi ﬁ cant pest due to the positive response in development when environmental scenarios have to change, speci ﬁ cally the increase in temperature.


Introduction
Sugarcane (Saccharum officinarum) is distributed between 36.7°north latitude and 31.0°southlatitude; that is, the cultivation of this species extends from tropical to subtropical zones [1].Currently, this species is the economic base of more than 100 countries, with more than 25 million hectares planted worldwide [2,3].The main characteristic that the sugarcane production system has presented is the continuous increase in the world production due to the increase in sugar and ethanol consumption [2], consolidating itself as an essential factor in the economy of many countries, maintaining its high demand in international trade [4,5], and registering a growth of 30% since the last 10 years [6].
Historically, in the Cauca River valley in Colombia, the leading sugarcane producing area for sugar and ethanol production, two species of the genus Diatraea (D. saccharalis and D. indigenella) had been identified as causing damage to sugarcane stalks.Biological control of these species is carried out with the release of tachinid flies (Lydella minense and Billaea claripalpis) and the genus Trichogramma [14][15][16].During 2012, the dominance of these two species was interrupted when outbreaks of D. tabernella and D. busckella occurred in the northern and central Cauca River valley [11,17].In 2013, Diatraea busckella caused significant damage to sugarcane crops in the center of the valley.D. busckella has a longer life cycle than D. saccharalis (about ten days more), causing more significant damage as it can open galleries of greater magnitude [18].Although D. busckella was reported in other parts of the country, it constitutes a first record for the region and is added to the other three known species [16].Currently, these species are in a critical situation for sugarcane producers [16].
In addition to changes in the behavior and increase in D. busckella population, this record may be a consequence of changes in different climatic variables, including air temperature, directly affecting arthropods in their biochemical processes [11].Since they cannot regulate their body temperature, they depend directly on environmental conditions to obtain the thermal units necessary to complete their life cycle [19,20].Consequently, as the air temperature increases, the development increases [21][22][23][24][25].However, some individuals can withstand temperatures above-known optima and achieve survival under previously unreported conditions.Because of this, the developmental rates of an insect are mainly based on the accumulation of thermal units measured in terms of physiological time [26], based on the calculation of the accumulation of degree days, thus defining the speed of development [27].
With the appearance of new records of arthropod pests that cause damage to crops due to possible changes in climate dynamics and supply in the region, it is necessary to seek an approach by monitoring and analyzing life cycles through a thermophysiological study that simulates an altitudinal profile where sugarcane can be planted.

Methods
This work was carried out at the Center for Research and Breeding of Natural Enemies, located in the botanical garden of the University of Caldas at 5.056388 north latitude and -75.494166 west longitude; at an altitude of 2,160 m above the sea level; and at an average temperature of 14°C (Manizales, Caldas).The laboratory work has its basis on two moments.
We started with 1,500 eggs for each treatment at five temperatures (10, 20, 24.7, 30, and 35°C), checking the time of hatching daily.The larvae were evaluated in 10 glass Petri dishes at a rate of 10 larvae per dish (100 replicates/temperature), maintaining optimal conditions that allowed the larvae's normal development to avoid deaths, pathogens, dehydration, or lack of food.
For the proper identification of the insect's stage, microscopic and macroscopic photos were taken daily.The analysis of photographs also determines the moments of change, as long as they were still alive.The Petri dishes were all-time closed to avoid the escape of larvae.It was in the same manner for the containers where the pupae and adults were kept.Pupae were placed inside a 30 cm × 25 cm × 12 cm emergency plastic container, covered with a 15 cm × 15 cm square of muslin cloth to facilitate aeration and covered with a perforated container lid.
After the change from larva to pupae, these were sexed and placed in separate containers for the adults to emerge, observing the last abdominal segment, where the external genital structures are for males, a bulge separated by a line (Figure 1a), and for females a line without bulge (Figure 1b).
Emerging adults were placed in plastic containers covered with a white paper in a 3:1 ratio (male:female) to obtain postures.Daily observations allowed to count eggs (when present) and identify the presence of pathogens.Sugar water in a moistened absorbent cotton was used for feeding, which was changed every 2 days.
The effect of temperature on the size and the growth rate of the cephalic capsule and the body length was evaluated with a generalized linear mixed model (GLMM) to differentiate the rate of change between each instar evaluated.The temperature treatments were the fixed factor, and the Petri boxes with the individuals per instar were the random factor.The packages mgcv, lme4, MASS, lmerTest, and pbktest were used for statistical analyses, performed in R version 1.1.414(R Development Core Team, 2009-2018).

Calculation of physiological time
The calculation of degree days (°D) was measured inside the growth chambers, using simple sine to simulate the hourly behavior of air temperature in a day using the daily maximum and minimum temperature as inputs (Figure 2) [27].These temperatures control the speed of development of many organisms that require a certain amount of heat to develop their life cycle [27,33].
For D. busckella, the equation describing the physiological behavior when the temperatures evaluated are between the maximum and minimum thresholds was used by applying to 24-hour periods [27].

Life cycle development of D. busckella
The number of days of the larval stage was 40 days DS ± 16.7 ± 2 days from larva to pupae and 6 ± 1 more to reach the adult stage under controlled conditions under five different temperatures (10, 20, 24.7, 30, and 35°C).The life cycle from egg to adult under laboratory conditions averaged 53 days ± SD 19, where at higher temperatures (30°C), the life cycle can be suspended due to adverse conditions (Table 1).
There is a change in the development rate, specifically from two larvae at 30°C, specifically from instar five to pupae, without passing through instar 6.It was concluded that all 6 instars are completed at low temperatures, increasing the total number of the cycle up to 72 days.
Therefore, the accumulation of degree days is lower, especially in instar 2 and 3.

Cephalic capsule size
The size of the cephalic capsule in the individuals evaluated indicates differences in size in 5 of the 6 larval   instars under different temperatures, where the increase is associated with the highest temperatures, following a behavior similar to the changes in the body length (Table 2).
The difference in the size of the cephalic capsule of D. busckella is greater between instars as the temperature increases.For the first two instars, the differences were 50.2 (30°C), 44.8 (20°C), and 48.95 (24.7°C), in response to environmental changes such as an increase in temperature (Table 3).

Length of D. busckella larvae
The GLMM model for the larval length showed highly significant values in the six larval stages, pupae, and adults in the three temperatures evaluated (Table 4), where growth is exponential in larval stages, with growth rates higher than 1 mm day −1 .They lose size when they reach the pupal stage due to metamorphosis processes (Table 4).
The larvae of D. busckella went through six instars before reaching pupa, with an average length of 30 mm under high temperatures in their last larval stage, higher values according to what has been reported by some authors in different species of Diatraea, indicating the direct relationship of the environment with their development.However, it should be taken into account that the results obtained were under laboratory conditions and could slightly affect the results under natural conditions.The differences in body length between instars (Table 5) are highly significant when the direct effect is temperature, where the largest sizes were found at 30°C and are well above the average temperature where D. busckella has been found (24.7°C).

Calculation of thermal constants and thresholds
For D. busckella, the maximum threshold temperature (30°C) and the optimum temperature (24.7°C) were used to calculate the accumulation of thermal time using the simple sine.The thermal time required to complete a cycle from egg to adult in D. busckella was defined as 660°D.
Biology and life cycle of Diatraea busckella (Lepidoptera: Crambidae)  929 accumulation of degree days is zero (0), occur at 10°C, stopping its entire physiological process, entering a dormant phase until its death (Figure 3).The rate of development follows an increase up to 30°C, where temperature, as a limiting factor in the development of organisms, conditions the presence or absence of D. busckella in some regions (Figure 3).Furthermore, these positive responses to temperature justify the growth of the insect, where the higher the temperature, the larger the size.Like other living organisms, insects can survive only within certain limits set by environmental factors such as temperature, relative humidity, and photoperiod.

Discussion
In the life cycles, a significant effect of temperature on development was observed, validating the results of ref. [11] who found variation in the number of instars associated with different thermal conditions in D. saccharalis.On the other hand, [11,27,29,[34][35][36] reported a record of up to 14 instars in overwintering larvae in D. saccharalis.By determining the total number of degree-days required for D. busckella to complete its entire life cycle, projections can be generated to simulate dates and duration times under different conditions.
These temperature effects are in contrast to Comadia redtenbacheri (Hamm) (Lepidoptera: Cossidae), where seven well-defined larval instars with changes in cephalic capsule size were presented about temperature effects [37].The progression and relationship between body length and cephalic capsule size between instars are regular [38,39].
Arthropods have a rigid exoskeleton, which is secreted by the epidermis.This makes it impossible for animals to gradually increase in size [40].Instead, the increase in body size is accomplished in stages, associated with the loss of the previous exoskeleton and the deposition of a new, larger one.This process is known as molting or ecdysis.The stages between molts are called instars and the overall size increase between molts can be remarkable [24,41].A salient feature of the hormones that stimulate metamorphosis is that, as well as influencing various tissues, they affect them in different ways, from the subtle to Signif.codes: 0, "***" -0.001, "**" -0.01, "*" -0.05, " ‡" -1. the overt [42][43][44][45].The higher temperature, the greater the growth in length.In its size changes, specifically in its last instar, the larva is affected by the secretion of prothoracicotropic hormone (PTTH); this acts on the prothoracicotropic gland stimulating the secretion of ecdysone, the hormone that overcomes the inhibition of the juvenile hormone and causes metamorphosis.There are alterations in the expression of many genesseveral hundred at leastduring metamorphosis; due to special features of polytene chromosomes, it is possible to observe some changes in gene activity taking place during metamorphosis [11,29,40,46].
Ecdysone is what promotes metamorphosis; however, its action can be counteracted by the juvenile hormone produced by the winged body, an endocrine gland located immediately behind the brain.As its name implies, it maintains the larval stage.In Lepidoptera, a pulse of ecdysone in the last larval instar triggers pupal formation, and another pulse a few days later initiates the late stages of metamorphosis [40,46,47].This differential sensitivity to different types of environmental stimuli provides a mechanism by which caterpillars should be able to respond simultaneously to multiple types of environmental variation.Final larval size is the result of a balance between these sensitivities and their responses [25,41].Temperature ranges also influence the level of response of activities such as feeding, dispersal, and laying, or it can be observed how at the lower thermal limit, the curve asymptotically approaches the zero point of development since insects tend to survive for long periods at low temperatures with slow development.Hence, the temperature at which initial development occurs (minimum developmental threshold) is difficult to measure accurately.
As the temperatures increase from this lower limit, the rate of development increases, and the function can be adjusted to a straight line in the intermediate zone.We approach the optimum temperature (that at which the rate of development is maximal).
A direct effect of temperature on the change in size is noted.When a larva has reached a particular instar, it does not grow or undergo further molting but is subjected to a more radical metamorphosis to the adult form.In insects, environmental cues (e.g., nutrition, temperature, and light), as well as the insect's internal developmental metabolism, affect the larva's central nervous system, emitting signals that act on a neurosecretory release site in the brain, secreting a prothoracicotropic hormone, stimulating ecdysone production [40,46].
The pupae of D. busckella, in females, presented a length of 17 mm and in males of approximately 15 mm, caused by size differences typical of dimorphism in Lepidoptera [47], while some authors have reported pupal lengths between 18 and 26 mm for other Diatraea species [11,29,35].The adult D. busckella had a length of 13 mm in males and a length of 16 mm in females.Temperature is considered to be the most important environmental factor influencing the performance of ectotherms, as it determines the rate of most biochemical reactions and thus the efficiency of metabolism and function [46,48,49].Adults lived between 4 and 5 days, in comparison with D. indigenella adults, which presented a length of 25-30 mm.In addition, the size of adults that was able to be determined in laboratory conditions was 2.1 cm [11,29,35,50,51].
The linear model has been one of the most important statistical tools for the user at the applied level.Its interpretability and the availability of computational results, together with the theoretical results, have made this model a very popular technique.To obtain estimates with this model, one of the fundamental requirements is the independence of the sample observations, with the use of Anova, it is possible to describe the generalized impact of temperature on the development of individuals, but with GLMM, it is possible to specify even the difference in size for each instar change according to the evaluated temperature.
Studying biology and reproductive development of insects in the laboratory at different temperatures helps to determine the effects on numerous biological parameters such as longevity, fecundity, sex ratio, development times, and degree-day requirements [20,52,53].The results allow inferring the tolerance that D. busckella has to temperature increases, so it can become a potential pest in environments different from those reported in Caldas where the average temperature is between 17 and 25°C since it was able to achieve survival and development under the evaluated scenarios of higher temperatures.Since is a tropical species, it does not have the mechanisms to remain in this lethargic state for a long time.When temperatures are higher than 35°C, they are considered extreme, since the organisms are subjected to stress conditions, which inhibit their development and result in an excessive accumulation of degree days, to the point of causing death (Figure 3); in contrast, temperatures below 10°C and above 35°C suppress its development and activity [36,54,55].As a result, development begins to slow down and then drops sharply.In addition, at temperatures above optimum, mortality rates are very high, which also makes it difficult to study the development at high temperatures and, therefore, to determine the maximum developmental threshold [19,21].
There are still no references showing the use of a generalized linear mixed model to compare the effect of temperature on the development of insect stages, so it could be used in different studies to corroborate the effectiveness of the model for more accurate interpretations.
The GLMM methodology allowed validation of the impact of temperature at each specific time.
It can be assumed that this organism has a possible adaptive capacity to remain in places where temperatures are above the current known temperatures for its development, considering the future potential of this organism to be a limiting pest and perhaps the main pest in sugarcane-producing areas.

Conclusion
The concept of degree days has been successfully used in the prediction of the development of different organisms, being a valuable tool for the management not only of insects but also of crops, since it helps producers and consultants to anticipate the resulting biological events.As a result, integrated crop management becomes more environmentally responsible, and better decisions are made to solve the problems that arise.
D. busckella can become a potential pest in other sugarcane-producing areas in Colombia, specifically in those where the temperature range is between 20 and 30°C where full development, reproduction, establishment, and survival are achieved.
The time it takes for an organism to complete its biological cycle is directly dependent on temperature.In the case of D. busckella, there is a direct relationship with development since metamorphosis takes place when inhibition, which predominates in the larval stage, is overcome in response to environmental signals.The signals generated by the two groups of endocrine cells control the development of all cells involved in metamorphosis since metamorphosis is largely affected depending on the changes that may occur.Thus, under global warming scenarios and, seeing the response of D. busckella to temperature changes, its colonization to new sites where no pest attack has been reported may be highly probable.

Figure 2 :
Figure 2: Simple sinus, entirely between the two thresholds (T U = upper limit, T L = lower threshold, T max = maximum temperature, T min = minimum temperature).Source: 27.Methodology for calculating heat Units designed by the University of California, Agriculture and Natural Resources, used in SIMARBC.Retrieved from http://www.simarbc.gob.mx/descargas/MetodologiaUC.pdf.

Table 1 :
Life cycle length (N°of total days/stage) of D. busckella at different temperatures under controlled conditions Development stage (days) Treatment (°C)

Table 2 :
Size (mm) of the cephalic capsule of D. busckella at different temperatures under controlled conditions

Table 3 :
Differences between the size (mm) of the cephalic capsule of D. busckella at three different temperatures 30, 20, and 24.7°C (T30,

Table 4 :
Body length (mm) of D. busckella larvae at different developmental stages under three different temperatures under controlled conditions

Table 5 :
Differences in body length (mm) between each instar at different temperatures under controlled conditions for D. busckella