The development of Panstrongylus herreri under fluctuating environmental conditions

Abstract:

INTRODUCTION:

Panstrongylus herreri is a main Chagas disease vector, and its success as a vector stems from its ability to establish domiciliated colonies; we aimed to explore its biology and reproduction.

METHODS:

The average amount of blood ingested and the time from the beginning of a blood meal to the production of feces were recorded.

RESULTS:

Females exhibited a higher blood ingestion rate than males, but similar defecation times and frequencies were observed.

CONCLUSIONS:

Despite the detected decrease in oviposition rates, P. herreri’s potential as a Chagas disease vector in environments other than the Amazon forest cannot be discounted.

Keywords:
Panstrongylus herreri; Biology; Life cycle

Chagas disease, which is caused by the protozoan Trypanosoma cruzi, affects millions of people around the world. The disease can be transmitted by different routes, but the route involving insect vectors known as triatomines represents the primary means of infection in endemic countries¹. Generally, the insect defecates after it bites a vertebrate host and ingests blood, and the host can subsequently become infected when parasites in the feces enter its body through disrupted mucous membranes or through the skin².

The Triatominae subfamily is comprised of approximately 140 species, and 109 of these species, which belong to the Rhodnius, Triatoma, and Panstrongylus genera, are major members of this group³. These species tend to live in either sylvatic or urban areas, but evolutionary pressures might allow some species to migrate from sylvatic areas and to adapt to domestic environmental conditions. Furthermore, migration from one ecosystem to another might be related to the introduction of different T. cruzi strains to urban areas⁴. Panstrongylus herreri, also described as P. lignarius⁵, is primarily found in the Amazonian forests of Peru, Ecuador, and Brazil, and it is one of the main vectors of Chagas disease in Peru⁶. However, this triatomine has the ability to successfully establish domiciliated colonies⁷, thus demonstrating its great potential as a Chagas disease vector and reinforcing the need to study its biology and reproductive aspects under varied environmental conditions.

Since one of the main characteristics of triatomines is their ability to adapt to and develop in distinct geographical areas under either sylvatic or domestic conditions, a better understanding of their biology, behavior, and ability to acclimatize to different environmental conditions may reveal important strategies that can be used to control both the vector and the spread of disease.

To this end, we conducted experiments to assess biology, reproduction, and feeding behavior; to study the impact of fluctuating environmental and geographical conditions on the development of P. herreri, 80 nymphs were monitored from egg eclosion to the adult stage. Insects used in this study were kindly donated by São Paulo State University, Araraquara, São Paulo, Brazil; they were raised and maintained in small plastic flasks (6.0cm height × 7.0cm diameter) at the insectary of the Federal University of Triângulo Mineiro [Universidade Federal do Triângulo Mineiro (UFTM)] until the 4^th nymphal stage was reached. Older nymphs and adults were maintained in intermediate-sized flasks (7.5cm height × 11.0cm diameter). All triatomines were provided with a natural light cycle, fluctuating environmental temperatures (max: 31.03°C ± 0.81°C; min: 18.87°C ± 1.387°C), and humidity conditions (56.85% ± 25.66%) in Uberaba, Minas Gerais, Brazil (19° 44′ 52″ S, 47° 55′ 55″ W; 823m above sea level). The experiments were conducted from September 2013 to March 2014, and specimens were fed weekly on immobilized and anesthetized Swiss mice. The mice were obtained from the animal facility at the Parasitology Division of the UFTM in Uberaba, Minas Gerais, Brazil, and were anesthetized using sodium thiopental (40mg/kg via intraperitoneal injection).

After oviposition, eggs were collected and monitored until eclosion. Eighty nymphs were then divided into colonies, and the time required to reach the adult phase was recorded. The 1^st nymphal stage insects were fed 10 days after eclosion or molting (2^nd and 4^th nymphal stages), and were then monitored until the next molt. Insects at the 5^th nymphal stage were fed twice (10 and 20 days after molting). After these feedings, only nymphs that were able to develop into adults were considered viable, and nymphs at the 5^th stage were considered viable after the second feeding.

Various aspects associated with the feeding behavior of the insects were examined, including the average feeding duration, the amount of blood ingested, the elapsed time between a blood meal and excretion, and the frequency of feces per meal. Twenty adult insects (10 males and 10 females) that were fasted for 28 days were randomly selected from pre-existing colonies. The rate of blood ingestion was calculated for each individual by dividing the amount of blood ingested by the feeding duration. The triatomines were individually housed in plastic flasks, and were weighed before and after each blood meal using an analytic scale (UniBloc Shimadzu AUY220).

To evaluate the effect of fasting on egg production, oviposition was supervised during specific fasting times. Therefore, eight adult couples were randomly chosen, isolated, and fed only once, and oviposition was subsequently monitored daily over a 21-day period. Eggs were collected on days 7, 14, and 21 after oviposition, and were kept in different containers for analysis.

The normal distribution and homogeneous variance of each variable were tested, and the size of the groups warranted the use of a non-parametric Mann-Whitney test to compare the time of feeding, amount of blood ingested, and the blood ingestion rate. Moreover, Fisher’s exact test was used to address the differences between the percentage of males and females that produced feces. Since only eight couples were analyzed, a non-parametric Friedman test was used to compare oviposition between weeks. The results were expressed as mean ± standard error of the mean, and differences were considered statistically significant when p < 0.05 (5%). All analyses were performed using GraphPad Prism 5.0 software (San Diego, CA).

The mean time for eclosion was 19.09 ± 0.66 days (Table 1). The results indicated that the developmental time from one stage to the next (specifically the five stages between the 1^st nymphal stage and the adult stage) was slightly longer than that observed at the previous developmental stage (18.61 ± 0.91, 21.38 ± 0.80, 22.74 ± 0.82, 24.82 ± 0.90, and 34.91 ± 1.86 days), respectively (Table 1). Hence, P. herreri seems to be able to acclimatize and reach sexual maturity under environmental conditions that differ from their normal conditions.

TABLE 1
Time required to complete different developmental stages.

Developmental stage	Median ± SD (days)
Eggs-N1 (n = 80)	19.09 ± 0.66
N1-N2 (n = 70)	18.61 ± 0.91
N2-N3 (n = 45)	21.38 ± 0.80
N3-N4 (n = 38)	22.74 ± 0.82
N4-N5 (n = 28)	24.82 ± 0.90
N5-adult (n = 11)	34.91 ± 1.86
Total	141.29 ± 5.81

Feeding behavior was assessed because P. herreri individuals were able to reach the adult stage under local environmental conditions, and because blood meals are key to understanding disease transmission and triatomine survival. The results indicated that females required more time than males to complete blood meals, ingested larger amounts of blood, and had higher blood ingestion rates (Table 2). Furthermore, 80% of the adult males defecated within 19.27 ± 6.03 minutes after starting a blood meal at a rate of 0.9 ± 0.56 feces/meal (Table 2). In contrast, females excreted within 18.81 ± 7.41 minutes at a rate of 1.0 ± 0.00 feces/meal (Table 2).

TABLE 2
Feeding behavior, time post-meal, and frequency of defecation.

	Time of feeding (min)	Blood ingestion (mg)	Blood ingestion rate (mg/min)	Time of defecation (min)	Insects producing feces (%)	Frequency of defecation (%)
P. herreri (M)	18.73 ± 7.77*	97 ± 38.10*	5.67 ± 2.47*	19.27 ± 6.03	80.0	0.90 ± 0.56
P. herreri (F)	30.06 ± 10.34*	258.7 ± 76.22*	9.23 ± 3.10*	18.81 ± 7.41	100.0	1.0 ± 0.00

We then investigated the ability of P. herreri to lay eggs (Table 3). Our results indicated that 123, 83, and 40 eggs were laid during the first, second, and third weeks, respectively (Figure 1A), and the oviposition rate was 15.37 ± 11.96, 10.37 ± 4.59, and 5.00 ± 4.92 for the first, second, and third weeks, respectively (Figure 1B). Furthermore, daily oviposition was also verified, and the rates were 2.19 ± 1.70, 1.48 ± 0.65, and 0.71 ± 0.70 for the first, second, and third weeks, respectively (Figure 1C). Despite the fact that no statistical differences were identified, the >60% reduction in oviposition indicated a dramatic decrease between the 1^st and 3^rd weeks of the study.

TABLE 3
Weekly Oviposition of Panstrongylus herreri during the fasting period.

		1st week
Couple (number)	Number of eggs	Standard deviation	Daily oviposition	Standard deviation
1	6	11.96348611	0.857142857	1.709069445
2	7		1.000000000
3	9		1.285714286
4	5		0.714285714
5	36		5.142857143
6	18		2.571428571
7	11		1.571428571
8	31		4.428571429
Total	123		2.196428571
		2nd week
Couple (number)	Number of eggs	Standard deviation	Daily oviposition	Standard deviation
1	17	4.596194078	2.428571429	0.656599154
2	11		1.571428571
3	10		1.428571429
4	9		1.285714286
5	10		1.428571429
6	11		1.571428571
7	14		2.000000000
8	1		0.142857143
Total	83		1.482142857
		3rd week
Couple (number)	Number of eggs	Standard deviation	Daily oviposition	Standard deviation
1	11	4.928053803	1.571428571	0.704007686
2	2		0.285714286
3	6		0.857142857
4	6		0.857142857
5	2		0.285714286
6	0		0
7	0		0
8	13		1.857142857
Total	40		0.714285714

FIGURE 1
Oviposition of Panstrongylus herreri during the fasting period. After a blood meal, eight couples were separated into individual plastic flasks for three weeks as previously described. (A). Total number of eggs. (B). Eggs per couple/week. (C). Average eggs per couple per day. A Friedman test was used to compare the differences in oviposition between the weeks. The results were expressed as mean ± standard error of the mean.

In the present study, P. herreri was able to reach sexual maturity and feed, and individuals only required a slight increase in developmental time from one stage to the next under varying conditions. However, the ability of P. herreri to lay eggs was reduced during the fasting period, which suggests that the reproductive aspects of this triatomine species are highly sensitive to food shortages.

Data concerning the life cycle of P. herreri are scarce in the literature, but our results were similar to those observed in a previous study that used comparable temperature conditions ⁸. Silva and Silva⁸ demonstrated the impact of two distinct temperatures on the development of several triatomine species, including P. herreri, and they concluded that triatomines exposed to temperatures as high as 30°C exhibited a shortened life cycle compared to those exposed to a lower temperature (25°C). These results are meaningful given that other important species for vectorial transmission in Brazil such as T. brasiliensis, T. infestans, and T. pseudomaculata have life cycles of 119.7, 161.9, and 232.6 days, respectively⁹. Furthermore, T. sordida, which is considered the most important species for vectorial transmission in the Uberaba region, had a life cycle of 183 ± 36 days under conditions similar to those used in our study¹⁰. Therefore, the fact that P. herreri exhibited an intermediate life cycle reinforces its potential as a vector in areas that are distant from its regular ecological niche.

Of the factors that dictate the ability of triatomines to adapt to different environments and to transmit disease, their feeding behavior is perhaps one of the most important. The results demonstrated that females not only required more time to complete blood meals, but they also ingested higher amounts of blood than their male counterparts. Although no differences were detected between the sexes regarding the post-meal time of defecation, the proportion of females producing feces tended to be greater than males (not significantly different), thus suggesting that females might have a greater capacity to transmit disease than males. In this context, because both blood meals and defecation are essential to the transmission of Chagas disease, triatomines with longer feeding times (such as those of the females observed in our study) are of great epidemiological importance¹¹. However, the importance of triatomines with shorter feeding times cannot be disregarded¹².

Taken together, the results presented here indicated that P. herreri is able to develop and reach sexual maturity in distant geographical areas. Food availability had a large influence on the rate of oviposition, which suggests that food shortages may have a great impact on the reproductive biology of the species. Furthermore, because females ingested greater amounts of blood than males, it is reasonable to assume that they are potentially stronger vectors. Lastly, because this triatomine species was able to reach sexual maturity under fluctuating environmental conditions similar to those of distant geographical areas, the role of this insect as a Chagas disease vector in environments other than the Amazon forest cannot be overlooked.

Ethical aspects

All procedures were scrutinized and approved by the Institutional Animal Care and Use Committee of the Federal University of Triângulo Mineiro (Brazil) under protocol 307.

REFERENCES

• ¹
  World Health Organization (WHO). First WHO report on neglected tropical diseases: working to overcome the global impact of neglected tropical diseases. ed World Health Organization/WHO Press, 2010.
• ²
  Tyler KM, Engman DM. The life cycle of Trypanosoma cruzi revisited. Int J Parasitol. 2001;31(5-6):472-81.
• ³
  Schofield CJ, Galvao C. Classification, evolution, and species groups within the Triatominae. Acta Trop. 2009;110(2-3):88-100.
• ⁴
  Moncayo A, Silveira AC. Current epidemiological trends for Chagas disease in Latin America and future challenges in epidemiology, surveillance and health policy. Mem Inst Oswaldo Cruz. 2009;104 (Suppl 1):17-30.
• ⁵
  Marcilla A, Bargues MD, Abad-Franch F, Panzera F, Carcavallo RU, Noireau F, et al. Nuclear rDNA ITS-2 sequences reveal polyphyly of Panstrongylus species (Hemiptera: Reduviidae: Triatominae), vectors of Trypanosoma cruzi Infect Genet Evol. 2002;1(3):225-35.
• ⁶
  Dias JCP, Prata A, Schofield CJ. Doença de Chagas na Amazônia: esboço da situação atual e perspectivas de prevenção. Rev Soc Bras Med Trop. 2002;35(6):669-78.
• ⁷
  Patterson JS, Barbosa SE, Feliciangeli MD. On the genus Panstrongylus Berg 1879: evolution, ecology and epidemiological significance. Acta Trop. 2009;110(2-3):187-99.
• ⁸
  Silva IGD, Silva HHGD. Estudo comparado do ciclo evolutivo de triatomíneos a duas temperaturas. Mem Instit Oswaldo Cruz. 1989;84(Suppl 4):499-500.
• ⁹
  Guarneri AA, Pereira MH, Diotaiuti L. Influence of the blood meal source on the development of Triatoma infestans, Triatoma brasiliensis, Triatoma sordida, and Triatoma pseudomaculata (Heteroptera, Reduviidae). J Med Entomol. 2000;37(3):373-79.
• ¹⁰
  Pelli A, Da Silva MA, Sarmento FR, Martins E, Da Mata SA, Domingues MA, et al. Population parameters for Triatoma sordida Stal, 1859: the most frequent vector for Chagas disease in the Triangulo Mineiro (Heteroptera, Triatominae). Rev Soc Bras Med Trop. 2007;40(1):25-28.
• ¹¹
  Zeledon R, Rabinovich JE. Chagas' disease: an ecological appraisal with special emphasis on its insect vectors. Annu Rev Entomol. 1981;26:101-33.
• ¹²
  Zeledon R, Alvarado R, Jiron LF. Observations on the feeding and defecation patterns of three triatomine species (Hemiptera: Reduviidae). Acta Trop. 1977;34(1):65-77.

• This work was supported by the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Publication Dates

• Publication in this collection
  Jan-Feb 2017

History

• Received
  15 June 2016
• Accepted
  18 Oct 2016