Introduction

Cutaneous leishmaniasis (CL) is a zoonotic disease that has had an increase in cases in Latin America in recent decades, becoming a public health problem [1]. In 2020, Colombia reported the second highest number of cases in Latin America with a total of 6,161 and an incidence rate of 23.34 cases per 100,000 inhabitants [2].

In Colombia, 167 species of sand flies have been reported, with a wide distribution in most geographical areas of the country, of which 14 have been incriminated as vectors of Leishmania spp. [3–7]. Insects of the genus Pintomyia (previously classified as the genus Lutzomyia), townsendi series (Pi. longiflocosa, Pi. spinicrassa, Pi. torvida, Pi. townsendi, Pi. quasitownsendi, and Pi. youngi) have been frequently associated with cases of CL in mountainous areas of Colombia, Venezuela, and Costa Rica, mainly at elevations above 1,500 meters above sea level (m.a.s.l.), and in areas where there are ecological disturbances associated with the expansion of monocultures such as coffee [8–10].

Between 1990 and 2014, the highest number of cases of CL was reported in the sub-Andean geographical area of the Magdalena River Valley, amid 1,000 and 2,000 m.a.s.l., mainly in the states of Huila and Tolima, where the Pintomyia longiflocosa species [9,11] was considered the most likely potential vector in this area [12,13]. In these states, Pi. longiflocosa is dominant with abundances >90% in the municipalities of Neiva, Baraya, Rivera, Campoalegre, and Algeciras [14] and in the state of Tolima in the municipalities of Chaparral and San Antonio [14,15], as well as in Abrego, Norte de Santander [16]. Additionally, Pi. longiflocosa is a species that presents highly anthropophilic and endophagic behaviors that incriminate it as a vector of Leishmania sp [3,17]. In this sense, this sandfly has shown a high susceptibility, under laboratory conditions, to infection with Leishmania (V) braziliensis, the main etiological agent of CL and its natural infection with Le. (V) guyanensis [18,19].

Considering the high relevance of these sandflies in the transmission of CL, it is important to highlight the scarce information on fundamental aspects of their biology, ecology, and bionomy [18,19]. Some relevant studies show the finding of individuals at immature stages (larvae) of Lu. longipalpis and Mi. oswaldoi in Brazil [20] and of Mi. atroclavata and Mg. migonei, mainly on the Colombian Caribbean coast, describing the abiotic characteristics of the probable sites of oviposition and breeding sites [21].

For most Pintomyia species, the description and analysis of the parameters of spatio-temporal distribution, oviposition rates, hatching, survival-mortality, duration of immature stages, and other life cycle characteristics under uncontrolled conditions, are aspects not known in their entirety. The determination of these parameters in natural conditions will provide information that will allow estimates of their vector capacity and likely predict the times of greatest risk for contracting CL in endemic areas.

Previous studies show that for Pintomyia species, Pi. spinicrassa, Pi. quasitownsendi and Pi. youngi, under standardized laboratory conditions, the average duration of their life cycle corresponds to 75.47; 69.85; and 61.07 days, respectively [9]. With respect to Pi. longiflocosa, classified within this group for its taxonomic characteristics, an average duration of 93.8 days for its life cycle under laboratory conditions has been reported [18], making this the longest cycle reported for the group of species studied.

Other studies have shown that, in natural circumstances in the sub-Andean zone of the states of Tolima and Huila, Pi. longiflocosa presents a bimodal seasonal variation in the abundance of adults, showing two peaks: the first in the month of February and the second between the months of July and August. These patterns would be related to abiotic factors such as temperature, precipitation, and relative humidity, which fluctuate according to the climatic season [22–24] and, in turn, could have an influence on immature stages (eggs, larvae, and pupae), accelerating or slowing down their development (prolonged latency cycles) [23].

In order to provide knowledge about the biology of this species, taking into account its seasonality, the objective of this study was to establish the parameters of the life cycle of Pi. longiflocosa under semi-controlled conditions in a rural area of the municipality of Campoalegre, Huila, during two seasons of the year.

Methods

Study area

The study was carried out in the rural area of the municipality of Campoalegre, Huila, in the village "Venecia" located in mountainous territory on the western flank of the western mountain range, with coordinates 2°39′47"LN and 75°14′31"LW at an altitude of 1,618 m.a.s.l. (Fig 1). This area is characterized by an average temperature of 20°C and an annual rainfall of approximately 1,000 mm with a bimodal tendency, displaying two periods of high rainfall during the months of March and November [24].

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Fig 1. Geographical location of the rural area where the experiment was conducted.

a) landscape of the area, b) installation of the monitoring area, c) microscopic view of stage II larvae (20x). The map was obtained from the database of INSTITUTO GEOGRÁFICO AGUSTÍN CODAZZI—IGAC https://www.igac.gov.co/node/211, a free and open-source database (license: https://creativecommons.org/licenses/by/4.0/deed.es, CC BY 4.0).

https://doi.org/10.1371/journal.pntd.0011518.g001

According to the Holdrige classification, this zone, corresponds to a premontane humid forest ecosystem [24]. The main economic activity of the area is associated with the use of land for the agricultural production of various products, especially for the cultivation of coffee (Coffea arabica). This area was chosen because the presence of Pi. longiflocosa is abundant, and it is also part of the state (Huila) where cases of cutaneous and visceral leishmaniasis have been reported [13].

Results

The life cycles monitored under semi-controlled conditions in the field showed significant differences in some of the parameters evaluated. C1 presented an average duration of 134.9 days, a shorter time (P<0.005) compared to C2, which displayed a duration of 148.78 days.

The GC in C1 presented a duration of 8.47 days on average, while in C2, it increased to 11.42 days (P = 0.001). The development of immature stages, specifically in the larval stages, was more extensive for C2. This presented significant differences for larvae in stages II and III (P<0.001 and P = 0.008, respectively). The pupal stage in C1 presented a significantly longer time than C2 (P = 0.003) (Table 1). Similarly, the longevity of mature individuals (adults) was more extensive during C2 (P = 0.014).

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Table 1. Life cycle parameters and developmental attributes of Pi. longiflocosa in two seasonal life cycles under semi-controlled field conditions.

https://doi.org/10.1371/journal.pntd.0011518.t001

Regarding the number of individuals obtained in the F1 evaluated for C1 and C2, significant differences (P< 0.005), were found in the average number of individuals per parental female, specifically, during immature stages of development (larvae I, II, III, IV and pupae). When comparing the number of individuals per stage between the two cycles, it was observed that it was higher for C1 (Table 1), and significant differences were found in the stages of larval development: Larvae I (P = 0.003), Larvae II (P = 0.008), Larvae III (P = 0.005), Larvae IV (P = 0.008), and pupae (P = 0.031). However, no differences were observed in the final number of adult individuals (P>0.05) (Table 1).

In relation to reproductive indicators, the results in C2 show that F = 75.72%/female, and PF = 26.03 eggs/female, were higher than those found for C1 (F = 47.52%/female and PF = 20 eggs/female), with P = 0.016 and P = 0.006, respectively; however, RF showed an increase for C1 females (RF = 9.37/female), contrary to that presented for C2, (RF = 5.02/female) (Table 1).

Referring to attributes of development, the productivity, hatching, and pupation were higher in C1. The greatest difference was evidenced in the data obtained for hatching, in which it was 0.68 during C1 and for C2, it was 0.76 (Table 1). The emergence of the adult was the highest attribute in both cycles, presenting a higher value during C2.

Differences were observed in the survival presented between the two cycles monitored. For C1, all stages showed survival values greater than 70%, while for C2, the values were lower, starting with a survival percentage of 58% in the egg stage and increasing in each stage until reaching values greater than 90% from larvae III.

The mortality rate evidenced in C1 for the different stages was 32% (egg), 4% (larvae I), 6% (larvae II), 6% (larvae III), 7% (larvae IV) and 4% (pupae). For C2, the highest mortality rates occurred during the egg stages: egg, larvae I, and larvae II (24%, 41%, 27%, respectively) compared to later developmental stages like larvae III, larvae IV, and pupae (10%, 9%, and 3%).

On the other hand, the climatic variables recorded (precipitation, temperature, and relative humidity) fluctuated significantly during the development of the cycles evaluated. In the study area, the total rainfall was 1082 mm (min: 38.4mm, max: 164.6mm), evidencing a bimodal behavior defined by an increase during the month of March 2020 and those from November to January 2021. The average temperature was 18.76°C (min:17.94°C; max: 19.59°C); the increase in temperature coincided with the dry seasons presented in the month of February and those between August and October of the year 2020. Finally, the average relative humidity was 88.30% (min: 78.91%, max: 95.62%), which decreased considerably in the months of February, April, and those between August and October of the year 2020. Consequently, for C1, the number of average individuals per stage of development was positively correlated with temperature (R = 0.79, P = 0.024), and precipitation levels (R = 0.84, P = 0.012), while the average number of individuals of C2, presented a negative association with precipitation (R = -0.73, P = 0.033).

The larval development obtained in C1 did not present significant correlations with the environmental variables evaluated (temperature: R = -0.071, P>0.05, humidity: R = -0.32, P>0.05 and precipitation: R = -0.6, P>0.05); however, the survival rate of individuals revealed a negative association with the average precipitation levels (Fig 2A).

On the other hand, the larval development of C2 presented a positive and significant correlation between the survival rate and the average precipitation levels (R = 0.79, P = 0.03) and a probable association with humidity (R = 0.75, P = 0.05). Finally, it showed a negative but not significant association with temperature (R = -0.68, P>0.05) (Fig 2B).

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Fig 2. Survival rate of immature stages and related environmental variables for Cycle 1(A) and Cycle 2 (B).

https://doi.org/10.1371/journal.pntd.0011518.g002

Discussion

In this study, carried out in the rural area of Campoalegre, Huila, Colombia under semi-controlled conditions, it was possible to monitor the development of Pi. longiflocosa during two periods of the year, which together covered a full year, allowing to follow the development of two consecutive life cycles, starting from two cohorts of individuals of this species. Similarly, it was feasible to evaluate the incidence of precipitation, temperature, and relative humidity variables on the life cycle of Pi. longiflocosa.

Although the sample size can be estimated as small for a more general analysis, considering that there is no specific information that indicates whether the species Pi. longiflocosa is classified as rare, compared to other sandfly species, and its distribution is restricted to specific areas of Colombia, the proposed sample met the needs of the analyzes used.

Given the great difficulty of monitoring the development of these insects in their natural habitats, the possibility of evaluating how the conditions of the natural environment are determinants for the populations of this type of insects and, therefore, responsible for the prevalence of diseases such as leishmaniasis, is usually lost. In this work, although sandflies were protected in breeding chambers from predators, direct rain, wind, and others, environmental variables were not controlled. Consequently, the results obtained from life cycle parameters may represent an approximation to the conditions of development of Pi. longiflocosa in its natural habitat.

Since the first investigations in sandflies, the difficulty of detecting breeding sites has been evidenced due to several factors, namely a) the small size of the larvae, b) the great variety of possible sites for the laying of eggs and for the development of the different stages, and c) areas with high richness of decomposing organic matter, where there are diverse interactions of these insects with multiple tree species, generating countless microhabitats available for oviposition and development of pre-reproductive stages [21].

By virtue of the above, in recent decades, their colonization in controlled laboratory conditions has been proposed as the main strategy to clarify important aspects of their biology, life cycle, bionomy, genetics, physiology, and transmission of pathogens as well as for the evaluation of insecticides and new vector control methods [33–35]. Despite advances in sandfly colonization methods and strategies, only a little more than 20 of the approximately 1,000 species of sandflies reported have been colonized and kept by several generations in the laboratory. The main problems, associated with the difficulty of establishing colonies in the laboratory, are related to non-adaptation to laboratory conditions, which is reflected in the low reproduction rate of insects [35,36], which makes this the main impediment to achieving a better knowledge of these species of interest in the aforementioned aspects. For this reason, studies related to the establishment and analysis of the life cycle of vector species of the genus Pintomyia are scarce [19].

In particular, for Pi. longiflocosa, a single laboratory life cycle study conducted by Neira et al. in 1998 [18] is known, which shows the great difficulty of maintaining colonies of this species in the laboratory. In this study, they reported an average duration of the life cycle from egg to adult of 93.8 days, a value much lower than that obtained in this research. The differences found can be due to various causes, ranging from the type of substrate for growth of immature stages, the frequency and source of food, to the number of individuals per brood vessel, among other aspects [37]. However, given that environmental variables were not controlled in this study and insects were more exposed to environmental changes, it could be inferred that this exposure was the main cause of the differences found between the two studies. Similarly, Neira et al. [18] showed, as in the present research, that the longest development time was evidenced in larva stage IV. It is likely that the sandfly has the genetic capacity to extend this stage if the environmental conditions are not the most favorable because it must store enough energy to successfully develop the next stage (pupa), in which the individual does not feed and metamorphosis to adult. Similarly, it is plausible that some form of diapause has occurred, a phenomenon that has been reported in other Pintomyia species. Diapause is considered an optional strategy expressed in response to environmental disturbances or scarcity of energy resources [37].

In the countries of Colombia, Brazil, and Venezuela, the life cycle under controlled laboratory conditions has been described for some species of the genus Lutozmyia and Pintomyia involved in the transmission of the etiological agent of CL, including Pi. spinicrassa, Pi. quasitownsendi, Pi. youngi [9], Pi. shannoni [27], Ev. evandroi, and Lu. longipalpis [20], and Pi. Ovallesi [19]. Within the parameters estimated for these species, and according to the results obtained in this study, important differences were evidenced. Pi. longiflocosa displayed an average cycle length greater than 120 days compared to the 30-to-96-day fluctuation range established for the aforementioned species under controlled laboratory conditions. The hatching period of the eggs estimated in the previous studies takes place between 5 and 20 days, and the duration in the different larval stages occurs in periods between 15 and 70 days, obtaining the emergence of the adults between the following 7 to 14 days. These data contrast with those obtained in this study, in which Pi. longiflocosa presented a notable increase in the time required for each stage of development, taking up to 25 days for the hatching of eggs, 88 days in immature stages, and between 18 to 28 days for the emergence of adults after transformation into pupae (Table 1).

The reasons for these differences may be the same as those argued in this section in relation to the work of Neira et al. [18]; moreover, since these are species other than Pi. longiflocosa and, although they are phylogenetically close to each other, they may have enough genetic variations that cause different physical and behavioral responses to environmental changes.

In relation to the average GC obtained in this research, it fluctuated according to the cycle of the year evaluated (Table 1); however, it agrees with what was reported by Neira et al. [18] under laboratory conditions, with an average of 12.5 days. Studies related to GC are scarce, and even more so, when it is intended to determine the duration of the same in field conditions. Among the few studies, is relevant an investigation carried out in Brazil by Falcão et al. in 2017 [38], in relation to the duration of the GC of females of Lutzomyia cruzi. The procedure consisted of conducting two experiments with labeled, released, and subsequently recaptured females. In the first experiment of 172 released females, only two were recaptured five days after their release, without eggs or blood in the abdomen; while, in the second release, only one female was recaptured on the fourth day, which, two days later, laid eggs, suggesting a minimum GC of 6 days. The temperature during the experiments ranged between 17 and 33°C.

Regarding the production of individuals per female, Neira et al. [18] found that the average number of eggs obtained from Pi. longiflocosa under laboratory conditions was 27.6 eggs/female in contrast to the range obtained in this study with an average of 12 to 15 eggs/female (Table 1). It is likely that egg fertilization could be a determining factor in egg production probably due to early individualization and post-feeding confinement of females [27]. In 2021, Ribeiro Da Silva et al. [39] also reported that average egg production of individualized females was lower in the established colony of Lu. longipalpis than that of females who remained in groups (31.4 and 35.7, respectively).

In this study, fecundity showed values below 10, and in C2, the value was very low (5.02). These results are much lower than other studies that analyzed this parameter. In 2021, Ribeiro da Silva et al. [39] found an average fecundity of 33 in Lu. longipalpis; Escovar et al. [34] with Pi. spinicrassa, determined under laboratory conditions, a real fecundity of 36 and a potential fecundity of 37.4; Souza et al. in 2009 [36], with the complex of cryptic species of Lu. longipalpis, found fecundity values between 26.3 and 41.5, and Justiniano et al., in 2004 [40] with Ny. umbratilis, fecundity from 23.6 to 26.1.

Regarding fertility, in this research, important differences were evidenced between the two cycles monitored, finding the highest value in C2 with 75.72%, a value closer to that obtained by Neira et al. [18], who reported a fertility percentage of 82.7%, for Pi. longiflocosa. Other studies show even closer values of 75.5% [34] and 77% [40]. These differences in fecundity and fertility parameters between species may represent genetic differences in loci that control biological characteristics, given by geographical, reproductive isolation or mutational randomness [37,39].

The estimated developmental attributes for Pi. longiflocosa showed values higher than 0.5 in both cycles—0.77 for C1 and 0.57 for C2. Statistical analyses showed significant differences between the two cycles. Hatching was higher in C2, and pupation and productivity attributes (from adults) were higher in C1.

Some studies evaluating adult productivity of Lutzomyia in the laboratory show results with high variability. With Lu. longipalpis, in 2000, Luitgards-Moura et al. [41] found a minimum productivity of 21.1 in F1 and a maximum of 67.2 in F2. Ribeiro Da Silva et al. [39], determined productivity with values close to 25%. In this study, productivity was 57% in C2, and 77% in C1. These are high productivity values compared to the mentioned studies. It is likely that the differences presented are due to the fact that the uncontrolled conditions of this study allowed a better expression of the biological efficacy of these insects [42]. Similarly, some of the disparities may be related to variations in the way this parameter is measured.

The lower survival rate of individuals for cycles C1 and C2 was evidenced during the immature stages, specifically in the egg stage, coinciding with what was evidenced for the Pintomyia species Pi. spinicrassa, Pi. quasitownsendi, and Pi. youngi- [10]. On the other hand, Neira et al. [18] found in Pi. longiflocosa that the highest percentage of mortality occurred in larva IV, arguing that the natural extension of this stage increases the probability of fungal contamination and predation by mites. However, laboratory studies with other species of Pintomyia show varied results in the stages with higher mortality; in larva I [34], egg and larva I [40], and egg and pupa [20]. In this sense, larval mortality is considered one of the main problems for the colonization of sandflies in the laboratory and, in particular, in the first stage, in which, due to their small size and low strength, individuals can be trapped in mycelia or in moisture condensation [35]. In this study, a positive correlation between precipitation, relative humidity and larval survival was found in C2, which probably explains the higher mortality of the Larva 1 stage, given that precipitation and relative humidity were lower in the second cycle. In contrast, Ribeiro Da Silva et al. [39], obtained a low mortality in pupae of Lu. longipalpis, arguing that the reason may be due to the fact that, at this stage, they do not feed and prefer dry environments in which invasion by fungi and other microorganisms is reduced. On the other hand, mortality can also be due to aspects related to microenvironment conditions, the difficulty of obtaining and assimilating nutrients and particular characteristics of the species itself.

The life cycles established for Pi. longiflocosa in the study area, covered seasonal periods associated with the fluctuation of precipitation in the Andean zone of Colombia. According to the correlations obtained, the survival of individuals could be positively associated with the increase in temperature and negatively with precipitation values (Figs 1 and 2). For the two cycles monitored in this study, the hatching of adults coincided with the low rainfall season (the month of August and those from December to February); and, as reported by Ferro et al [16], the number of adults of this species presents an increase in its abundance marked by the decrease in rainfall. Similarly, Rodríguez and Carvajal (2015) [12,43] also point out a negative association between the abundance of individuals and precipitation, based on a study carried out in 56 microhabitats located in the sub-Andean region of the upper Magdalena Valley between 100 and 2002 m.a.s.l., altitudinal conditions similar to those of this work.

Moreover, for 5 years Valderrama et al. [43], evaluated different localities at medium elevations of the Colombian Andean region (1,000 to 2,000 m.a.s.l.) climatic variables as possible risk factors for the incidence of cutaneous leishmaniasis, associated with the presence of Pintomyia species, concluding that the peak incidence of the disease is associated with a mean temperature of 20.6°C (95% CI = 19.2–22.0°C), a factor that, in turn, would be related to other variables such as vegetation cover and land use [11]. It should be noted that the average temperature recorded in this study was 18.76° C, very close to that reported in the aforementioned study, which allows us to infer a relationship between the abundance of Pi. longiflocosa and temperature. Consequently, the temporal distribution of this possible vector in areas of endemic transmission of CL would be conditioned by specific parameters of its life cycle in relation to the variation of the abiotic factors of their habitats.

The variations in population parameters, production attributes and duration of the life cycle found between the two cycles monitored in this study may indicate the effect of environmental variables on the development of the biological cycle of Pi. longiflocosa in its natural habitat.

The information obtained in this study contributes to the knowledge of the reproductive dynamics of Pi. longiflocosa in semi-controlled environmental conditions, which can more reliably represent the behavior of this vector in nature; however, it is recommended to include other reproductive parameters in future studies, with a larger sample and more monitoring time.

Supporting information

Acknowledgments

To Marco Fidel Suárez for his valuable management and dedication in the development of the field phase of this project.