Life cycle and reproductive patterns of Triatoma rubrovaria ( Blanchard , 1843 ) ( Hemiptera : Reduviidae ) under constant and fluctuating conditions of temperature and humidity

The aim of this study was to evaluate the temperature and relative humidity influence in the life cycle, mortality and fecundity patterns of Triatoma rubrovaria. Four cohorts with 60 recently laid eggs each were conformed. The cohorts were divided into two groups. In the controlled conditions group insects were maintained in a dark climatic chamber under constant temperature and humidity, whereas triatomines of the ambiental temperature group were maintained at room temperature. Average incubation time was 15.6 days in the controlled conditions group and 19.1 days in the ambiental temperature. In group controlled conditions the time from egg to adult development lasted 10 months while group ambiental temperature took four months longer. Egg eclosion rate was 99.1% and 98.3% in controlled conditions and ambiental temperature, respectively. Total nymphal mortality in controlled conditions was 52.6% whereas in ambiental temperature was 51.8%. Mean number of eggs/ female was 817.6 controlled conditions and 837.1 ambiental temperature. Fluctuating temperature and humidity promoted changes in the life cycle duration and in the reproductive performance of this species, although not in the species mortality. Key-words. Life cycle. Reproductive characteristics. Temperature. Humidity. Triatoma rubrovaria. RESUMO O presente trabalho teve como objetivo avaliar a influência da temperatura e umidade relativa sobre o ciclo biológico, mortalidade e fecundidade de Triatoma rubrovaria. Formaram-se quatro coortes, cada uma de 60 ovos recentemente colocados. As coortes foram divididas em dois grupos: O grupo condições controlada foi mantido em estufa com temperatura e umidade constantes, e o grupo temperatura ambiental foi mantido em condições variáveis de temperatura e umidade. O tempo de incubação dos ovos foi de 15.6 condições controlada e de 19.1 dias temperatura ambiental. O período de desenvolvimento de ovo a adulto foi de dez meses em condições controlada e 4 meses mais extenso em temperatura ambiental. A taxa de eclosão foi 99.1% em condições controlada e 98.3% em temperatura ambiental. A fecundidade foi 817.6 condições controlada e 837.1 temperatura ambiental. O percentual de mortalidade ninfal foi 52.6% no grupo condições controlada e 51.8% no grupo temperatura ambiental. As variações de temperatura e umidade relativa exercem influência no ciclo biológico e em alguns padrões reprodutivos desta espécie, mas não em sua mortalidade. Palavras-chaves: Ciclo biológico. Padrões reprodutivos. Temperatura. Umidade. Triatoma rubrovaria. 1. Departamento de Biología, Facultad de Ciencias Exactas y Naturales y Agrimensura, Universidad Nacional del Nordeste, Corrientes, Argentina. 2. CRILAR – CONICET, Anillaco, La Rioja, Argentina. Address to: Lic. Miryam P. Damborsky. Cátedra de Artrópodos, Facultad de Ciencias Exactas y Naturales y Agrimensura. Universidad Nacional del Nordeste. Av da Libertad 5470, (3400) Corrientes, Argentina. Fax: 54 37 8347-3930. e-mail: mdambor@exa.unne.edu.ar Recebido em 16/11/2004 Aceito em 6/6/2005 Triatoma rubrovaria (Blanchard, 1843) is widely distributed in the Argentina Mesopotamia, Uruguay and South of Brazil. Its presence in the province of Corrientes (Argentina) was verified in basaltic outcrops of the Mercedes department. In Brazil it has been occasionally encountered in domestic and peridomestic environments in rural areas of the Paraná and Río Grande do Sul States, increasing its presence in human dwellings where Triatoma infestans has been controlled. T. rubrovaria

Triatoma rubrovaria (Blanchard, 1843) is widely distributed in the Argentina Mesopotamia, Uruguay and South of Brazil 8 .Its presence in the province of Corrientes (Argentina) was verified in basaltic outcrops of the Mercedes department 4 .
In Brazil it has been occasionally encountered in domestic and peridomestic environments in rural areas of the Paraná and Río Grande do Sul States, increasing its presence in human dwellings where Triatoma infestans has been controlled 1 .T. rubrovaria is a generalist species feeding from a wide variety of hosts including humans, and its competence as Trypanosoma cruzi vector has been experimentally verified 2 20 23 .
Higher temperature and lower relative humidity are climatic factors that decrease the development time from egg to adult of Chagas disease vectors 7 , promoting a higher population density, an increase in the feeding frequency and an expansion of the geographic distribution 9 10 21 .
The influence of temperature on the biology of Triatoma rubrovaria as well as in other Triatominae species has been reported 6 11 13 18 19 22 .Studies on cold resistance showed that T. rubrovaria was the most affected among a number of Triatominae species 5 .However, there is little information concerning the development of Chagas disease vectors under fluctuating temperature and relative humidity 15 .
The aim of the present study was to evaluate the temperature and relative humidity influence on the life cycle and mortality and reproductive patterns of T. rubrovaria.

MATERIAL AND METHODS
A total of 93 T. rubrovaria (6 N1; 15 N2; 17 N3; 25 N4 and 30 N5) were captured in an area of natural rock piles, located 37km from Mercedes City (29º 23' S, 57º 50' W) in the province of Corrientes, Argentina.The investigation was carried out with the first generation offspring from October 2000 to August 2003.
Four cohorts with 60 recently laid eggs (0-48 hours old) each were conformed.The eggs were kept in 60cm 3 plastic containers until all those that were viable hatched.The cohorts were divided into two groups: controled conditions (CC) and ambiental temperature (TA).The nymphs were transferred into 300cm 3 glass containers.The jars were covered with nylon mesh and provided with vertically placed strips of Whatman paper n° 4 to allow insects access to the food resource and for absorption of excess humidity.The specimens of both groups were fed weekly on chickens during 30 minutes.
The CC group insects were maintained in a dark climatic chamber under constant temperature and humidity.The triatomines of the TA group were maintained under room temperature and humidity with a photoperiod of 12L:12D hr.The temperature and humidity were measured with a Weatherlink-Monitor II -Davis weather station.
Adult couples were placed in individual containers to evaluate fecundity.Egg hatching was recorded daily and ecdysis, mortality, fecundity and fertility were noted weekly.
The rate of total nymphal mortality (TNM) was calculated as the ratio between the number of dead nymphs of all stadia (d t ) and the number of living nymphs at time t (N t ), and it was expressed as a percentage using the formula: TNM = d t /N t x 100.
The absolute nymphal mortality (ANM) was estimated as the ratio between the number of dead nymphs of a specific nymphal stage (d x ) and the total number of nymphs of that stage (N x ) x 100: ANM= d x /N x x 100.Longevity was measured as the time elapsed since each individual moulted to the adult stage until its death.
The fecundity was obtained as the ratio between all laid eggs and the total number of females.The fertility was calculated as the number of hatched eggs of the total number of laid eggs.
Statistical analysis.Analysis of variance (ANOVA) was used to compare developmental time, mortality, longevity, number of eggs/ female/week and number of reproductive weeks between groups.The variables were transformed to log (x+1) or hyperbolic arcsine.Post-hoc comparisons were made using the Duncan test.Male-female ratio was compared using chi-square test.Fecundity, fertility and first oviposition age between groups were statistically measured by the Kruskall-Wallis test.The test for difference of proportions was used to compare the egg survival between groups.Pearson coefficient (r) was used to analyze the correlation between monthly fecundity of the TA couples and monthly mean temperature.All data obtained were analyzed at a 5% significance level.Statistical analysis was carried out with the program INFOSTAT 2002, version 1.1 12 .
Triatoma rubrovaria of the TA group completed one egg to adult cycle from October 2000 to December 2001.During this period, the mean room temperature fluctuated between 22.1°a nd 28.2°C.In nine of these 15 months, the temperature was less than 26°C and the relative humidity lower than 56%.The lowest absolute temperature values (  1).Seventyfive percent of the eggs hatched between 15 and 16 days in the CC, while those in group TA took between 18 and 21 days.The mean development time of each nymphal stage was significantly different within the groups (F 5, 12 = 390.30,p< 0.0001), as well as between groups (F 1, 12 = 89.88,p<0.0001).The highest variation was observed in the third (N3), fourth (N4) and fifth nymphal development (N5) (p< 0.05).In group TA the fifth nymphal instars took 69 days longer than the CC group to reach the adult stage.In group CC the time from egg to adult development lasted 10 months while group TA took four months longer.
Nymphal recruitment differed by one week between groups until the third nymphal stage.From the fourth stage the recruitment rate was lower in group TA.Fifth stage recruitment took 16 weeks in CC group while group TA took 39 weeks.
In CC the fifth nymphal instars took 41 weeks to reach the adult stage, whereas in TA they moulted to adults in just 10 weeks.
The male to female ratio was 1:1.5 in CC and 1:1.2 in TA, no significant difference was observed (χ2 = 0.04, p= 0.8404).
The total nymphal mortality in CC was 52.6% ± 2.5 and in TA was 51.8% ± 7.9 (ANOVA, F 1, 10 = 0.08, p= 0.7790).The lowest mortality rate in CC group was observed in the second nymphal instars, and in TA in the fifth nymphal instars (Figure 3).Before moulting to the last nymphal stage, 33.3% nymphs died in CC, while 48.3% nymphs in the TA group did not reach this stage.
The highest mortality rate occurred during the fifth stage in group CC and during the third stage in group TA, when maximum room temperature reached 31.7°C and humidity 79%.Significantly different mortality between groups was noted in N1, N2 and N5 (Duncan test, p< 0.05).
Female age at first oviposition was 7.2 ± 2.8 days in group CC and 10.1 ± 4.6 days in TA (H = 21.94,p< 0.0001).
Mean number of eggs/female/week (ANOVA, F 1,101 = 4.43, p = 0.0378) and number of reproductive weeks (F 1,45 = 6.53, p = 0.0141) were different between groups.The highest number of eggs/female/week in TA was observed during hottest months.A direct correlation between fecundity and temperature was verified (r = 0.56, p< 0.001, n = 251).
An interruption period of up to six weeks in the oviposition occurred in all the TA females during cold as well as warm months.This interruption was only observed in 5 CC females and the longest duration was 3 weeks.After the interruption, the females continued oviposition until they died.

DISCUSSION
Most studies on life cycles in Triatominae have been carried out under controlled conditions of temperature and relative humidity, although these factors show daily and seasonal variations in natural habitats 15 .
Although no assessments of the natural biotope of T. rubrovaria have been carried out, studies on microclimatic conditions within a similar habitats of rock piles where T. brasiliensis is found, demonstrated that temperature and relative humidity reaches lower ranges than the external condition (especially on the rock surface) 14 .
Experimental studies revealed that higher temperatures cause a reduction in the developmental period of T. rubrovaria 22 .Higher temperature and lower relative humidity also decrease the development time of eggs and nymphs of T. rubrofasciata, T. melanosoma, R. neglectus and R. robustus 6 11 18 19 .Extremely low humidity can interrupt the insect development 15 , although no influence of humidity on egg development time of R. neglectus was found 18 .
Although average temperature and relative humidity were similar in the two experimental situations under which T. rubrovaria were reared, the variability was higher under the ambient conditions compared with the condition inside the climatic chamber.Room temperature was similar and relative humidity was slightly lower than records obtained within a palm trees community in the San Roque department of Corrientes 3 .
Development time in the TA group was longer than in the CC group, corroborating studies on the same species 22 and on other triatomines, describing longer life cycles under changing conditions of temperature and relative humidity 16 .
Adult emergence in the CC group occurred almost all year long (February to December 2001) at an approximately constant rate, whereas all nymphs of the TA group reached the adult stage between October and November 2001.In the last experimental group, no recruitment occurred during the colder months (April to September 2001).
Sex rate revealed a slight predominance of females in the two experimental groups, suggesting a higher mortality risk during the preadult development in males, as indicated for T. infestans 17 .Male longevity in the TA group was longer, coinciding with Triatoma rubrofasciata 6 .
Changing conditions of temperature and relative humidity did not affect egg survival and total nymphal mortality, as no difference was observed between the two experimental groups.However, the high mortality rate in the third stage in TA occurred during a period when the highest temperature and relative humidity were recorded.At 33°C and 70% a complete mortality for Rhodnius neglectus fifth nymphal instars was also reported 18 .
Fecundity and fertility did not differ greatly between the two experimental groups, coinciding with the results reported by Silva 22 when the species was kept at 25°C and 30°C.Date of first reproduction (from initial day of the study) began earlier in the CC group with higher variability, as the result of the long recruitment period to the adult stage in this group.
Constant temperature and humidity were the most optimal conditions for development, the life cycle of T. rubrovaria reared under these conditions was shorter.Although females under room conditions had a longer reproductive period, the number of eggs/ week was lower and oviposition interruptions were constantly observed.Developmental time in the group exposed to fluctuating temperature and relative humidity took over a year to complete.As the insects had no food restriction, this might be considered as the species minimum under natural climatic conditions.
This study indicates that fluctuating temperature and humidity have an important effect on the life cycle duration of T. rubrovaria.A difference of 2°C in the mean temperature under which the two experimental groups were developed, promoted changes in the life cycle duration and in the reproductive performance of this species.(69.7±18.9)SD: standard deviation.CC: controled conditions.TA: ambiental temperature.

Figure 1 -
Figure 1-Monthly mean room temperature from October 2000 to August 2003.

Figure 2 -
Figure 2 -Monthly mean ambient relative humidity from October 2000 to August 2003.
22.1°C and 23.6°C) were recorded from May to September 2001.Development time.The average incubation time was 15.6 days in CC group and 19.1 days in TA (Table