Efficacy of factory-treated and dip-it-yourself long lasting insecticide-treated bednets against cutaneous leishmaniasis vectors in the sub-Andean region of Colombia: results after two years of use.

BACKGROUND: Long lasting insecticide-treated nets (LLINs) may be effective for vector control of cutaneous leishmaniasis (CL). Their efficacy, however, has not been sufficiently evaluated. OBJECTIVE: To evaluate the large-scale efficacy of LLINs on Lutzomyia longiflocosa entomological parameters up to two years post-intervention in the sub-Andean region of Colombia. METHODS: A matched-triplet cluster-randomised study of 21 rural settlements, matched by pre-intervention L. longiflocosa indoor density was used to compare three interventions: dip it yourself (DIY) lambda-cyhalothrin LLIN, deltamethrin LLIN, and untreated nets (control). Sand fly indoor density, feeding success, and parity were recorded using CDC light trap collections at 1, 6, 12, and 24 months post-intervention. FINDINGS: Both LLINs reduced significantly (74-76%) the indoor density and the proportion of fully engorged sand flies up to two years post-intervention without differences between them. Residual lethal effects of both LLINs and the use of all nets remained high throughout the two-year evaluation period. CONCLUSIONS: Both LLINs demonstrated high efficacy against L. longiflocosa indoors. Therefore, the deployment of these LLINs could have a significant impact on the reduction of CL transmission in the sub-Andean region. The DIY lambda-cyhalothrin kit may be used to convert untreated nets to LLINs increasing coverage.

Experimental hut to study the indoor behaviour and effects of insecticide-treated bednets on phlebotomine sand flies (Diptera: Psychodidae).

INTRODUCTION: Behavioural effects of insecticides on endophagic phlebotomine sand fly vectors of Leishmania are poorly understood mainly because of the lack of an experimental hut (EH) in which to study them. OBJECTIVE: To build an EH to evaluate the effects of long-lasting insecticide-treated nets (LLINs) on Lutzomyia longiflocosa. METHODS: The study had two phases: (1) Laboratory experiments using tunnel tests to select the traps for the EH; and (2) EH construction and evaluation of the effects of deltamethrin and lambda-cyhalothrin LLINs on L. longiflocosa females inside the EH. FINDINGS: Phase 1: The horizontal-slit trap was the best trap. This trap collected the highest percentage of sand flies, and prevented them from escaping. Therefore, this trap was used in the EH. Phase 2: The main effects of LLINs on L. longiflocosa in the EH were: landing inhibition, inhibition from entering the bednet, induced exophily, and high mortality (total and inside exit traps). CONCLUSIONS: The EH was effective for evaluating the effects of LLINs on endophagic sand flies. Although both types of LLINs showed high efficacy, the lambda-cyhalothrin-treated LLIN performed better. This is the first report of induced exophily in sand flies.

Entering and exiting behaviour of the phlebotomine sand fly Lutzomyia longiflocosa (Diptera: Psychodidae) in rural houses of the sub-Andean region of Colombia.

The present study identified the entering and exiting sites for Lutzomyia longiflocosa in rural houses of the sub-Andean region in Colombia. Entering sites were identified with sticky traps set up outside the bedrooms, around the eave openings, and with cage traps enclosing the slits in the doors and windows inside the bedrooms. Exiting sites were identified by releasing groups of females indoors. These females were blood fed and marked with fluorescent powders. Females were recaptured with the trap placement described above but set up on the opposite sides of the openings. In the entering experiment, a significantly higher number of females were captured in the sticky traps at the zone nearest the eave openings (n = 142) than those captured in the other zones of the trap (n = 52); similarly, a higher number of females were captured on the front side of the house (n = 105) than at the rear side (n = 37). Only two females were collected in the cage trap. In the exiting experiment, at the ceiling, the highest percentage (86.2%) of females was recaptured with sticky traps nearest the eave openings and on the front side of the house (70.0%). Seven females were collected in the cage trap. Lu. longiflocosa entered and exited houses primarily through the eave openings in a non-random pattern in relation to the sides of the house.

Culicoides Latreille (Diptera, Ceratopogonidae) of Colombia: records from the collection of insects of medical importance from National Institute of Health.

Background: The collection of insects of medical importance from the Instituto Nacional de Salud, INS (Bogotá, Colombia: https://www.ins.gov.co/Paginas/Inicio.aspx), was started in 1934 with the aim of being an institutional and national repository of the biodiversity of insects involved in vector-borne diseases of importance in public health. Today, the entomological collection includes more than 7,500 specimens.The ceratopogonid insects are one group of Diptera that are represented in this collection. Within the Ceratopogonidae, the genus Culicoides Latreille, 1809 is relevant in public health because of the nuisance caused by their bites when they are presented in great abundance and because of their role as vectors of several agents (virus, protozoa and nematodes) that cause diseases to humans and to animals (Mellor et al. 2000, Mullen 2002). An overview of the Ceratopogonidae, represented in this collection, is presented here. A total of 801 individuals, mainly adults of the genus Culicoides (90%) are represented. The collection is the result of the effort of several researchers of the Group of Entomology at INS. These researchers collected ceratopogonids when they went to different transmission scenarios of vector-borne diseases in Colombia, with the purpose of making entomological characterisations including the processing, assembly and identification of the specimens in the laboratory. New information: New information about the geographical distribution of 39 species of the genus Culicoides in Colombia. All data have been uploaded to GBIF and are publicly available there.

Daily dataset of precipitation and temperature in the Department of Cauca, Colombia.

This study used the geostatistical Kriging methodology to reduce the spatial scale of a host of daily meteorological variables in the Department of Cauca (Colombia), namely, total precipitation and maximum, minimum, and average temperature. The objective was to supply a high-resolution database from 01/01/2015 to 31/12/2021 in order to support the climate component in a project led by the National Institute of Health (INS) named "Spatial Stratification of dengue based on the identification of risk factors: a pilot study in the Department of Cauca". The scaling process was applied to available databases from satellite information and reanalysis sources, specifically, CHIRPS (Climate Hazards Group InfraRed Precipitation with Station Data), ERA5-Land (European Centre for Medium-Range Weather Forecasts), and MSWX (Multi-Source Weather). The 0.1° resolution offered by both the MSWX and ERA5-Land databases and the 0.05° resolution found in CHIRPS, was successfully reduced to a scale of 0.01° across all variables. Statistical metrics such as Root Mean Squared Error (RMSE), Mean Absolute Error (MAE), Person Correlation Coefficient (r), and Mean Bias Error (MBE) were used to select the database that best estimated each variable. As a result, it was determined that the scaled ERA5-Land database yielded the best performance for precipitation and minimum daily temperature. On the other hand, the scaled MSWX database showed the best behavior for the other two variables of maximum temperature and daily average temperature. Additionally, using the scaled meteorological databases improved the performance of the regression models implemented by the INS for constructing a dengue early warning system.

Spatial Analysis of Dengue Clusters at Department, Municipality and Local Scales in the Southwest of Colombia, 2014-2019.

Dengue is an arbovirus transmitted by mosquitoes of the genus Aedes and is one of the 15 main public health problems in the world, including Colombia. Where limited financial resources create a problem for management, there is a need for the department to prioritize target areas for public health implementation. This study focuses on a spatio-temporal analysis to determine the targeted area to manage the public health problems related to dengue cases. To this end, three phases at three different scales were carried out. First, for the departmental scale, four risk clusters were identified in Cauca (RR ≥ 1.49) using the Poisson model, and three clusters were identified through Getis-Ord Gi* hotspots analysis; among them, Patía municipality presented significantly high incidence rates in the time window (2014-2018). Second, on the municipality scale, altitude and minimum temperature were observed to be more relevant than precipitation; considering posterior means, no spatial autocorrelation for the Markov Chain Monte Carlo was found (Moran test ˂ 1.0), and convergence was reached for b1-b105 with 20,000 iterations. Finally, on the local scale, a clustered pattern was observed for dengue cases distribution (nearest neighbour index, NNI = 0.202819) and the accumulated number of pupae (G = 0.70007). Two neighbourhoods showed higher concentrations of both epidemiological and entomological hotspots. In conclusion, the municipality of Patía is in an operational scenario of a high transmission of dengue.

Spatial patterns associated with the distribution of immature stages of Aedes aegypti in three dengue high-risk municipalities of Southwestern Colombia.

Aedes aegypti mosquitoes are the main vector of human arbovirosis in tropical and subtropical areas. Their adaptation to urban and rural environments generates infestations inside households. Therefore, entomological surveillance associated with spatio-temporal analysis is an innovative approach for vector control and dengue management. Here, our main aim was to inspect immature pupal stages in households belonging to municipalities at high risk of dengue in Cauca, Colombia, by implementing entomological indices and relating how they influence adult mosquitos' density. We provide novel data for the geographical distribution of 3,806 immature pupal stages of Ae. aegypti. We also report entomological indices and spatial characterization. Our results suggest that, for Ae. aegypti species, pupal productivity generates high densities of adult mosquitos in neighbouring households, evidencing seasonal behaviour. Our dataset is essential as it provides an innovative strategy for mitigating vector-borne diseases using vector spatial patterns. It also delineates the association between these vector spatial patterns, entomological indicators, and breeding sites in high-risk neighbourhoods.

Establishment and characterisation of a new cell line derived from Culex quinquefasciatus (Diptera: Culicidae).

Insect cell cultures are an important biotechnological tool for basic and applied studies. The objective of this work was to establish and characterise a new cell line from Culex quinquefasciatus embryonic tissues. Embryonated eggs were taken as a source of tissue to make explants that were seeded in L-15, Grace's, Grace's/L-15, MM/VP12, Schneider's and DMEM culture media with a pH range from 6.7-6.9 and incubated at 28ºC. The morphological, cytogenetic, biochemical and molecular characteristics of the cell cultures were examined by observing the cell shapes, obtaining the karyotypes, using a cellulose-acetate electrophoretic system and performing random amplified polymorphic DNA-polymerase chain reaction analysis, respectively. The Grace's/L-15 medium provided the optimal nutritional conditions for cell adhesion and proliferation. Approximately 40-60 days following the explant procedure, a confluent monolayer was formed. Cellular morphology in the primary cultures and the subcultures was heterogeneous, but in the monolayer the epithelioid morphology type predominated. A karyotype with a diploid number of six chromosomes (2n = 6) was observed. Isoenzymatic and molecular patterns of the mosquito cell cultures matched those obtained from the immature and adult forms of the same species. Eighteen subcultures were generated. These cell cultures potentially constitute a useful tool for use in biomedical applications.

Arbovirus vectors in municipalities with a high risk of dengue in Cauca, Southwestern Colombia.

The Culicidae family has two of the most important disease vector genus: Aedes spp. and Culex spp. Both of these are involved in the transmission of arboviruses. Here, we provide novel data for the geographical distribution of 2,383 specimens in the Culicidae family. We also report the percentage of houses infested with these vectors, and Shannon and Simpson diversity indices in three municipalities located in Cauca, Colombia. This dataset is relevant for research on vector-borne diseases because Aedes spp. can transmit arboviruses such as dengue, Zika and chikungunya, and Culex spp. is a well-known vector of West Nile virus and Venezuelan equine encephalitis.

Publishing data to support the fight against human vector-borne diseases.

Vector-borne diseases are responsible for more than 17% of human cases of infectious diseases. In most situations, effective control of debilitating and deadly vector-bone diseases (VBDs), such as malaria, dengue, chikungunya, yellow fever, Zika and Chagas requires up-to-date, robust and comprehensive information on the presence, diversity, ecology, bionomics and geographic spread of the organisms that carry and transmit the infectious agents. Huge gaps exist in the information related to these vectors, creating an essential need for campaigns to mobilise and share data. The publication of data papers is an effective tool for overcoming this challenge. These peer-reviewed articles provide scholarly credit for researchers whose vital work of assembling and publishing well-described, properly-formatted datasets often fails to receive appropriate recognition. To address this, GigaScience's sister journal GigaByte partnered with the Global Biodiversity Information Facility (GBIF) to publish a series of data papers, with support from the Special Programme for Research and Training in Tropical Diseases (TDR), hosted by the World Health Organisation (WHO). Here we outline the initial results of this targeted approach to sharing data and describe its importance for controlling VBDs and improving public health.

Breeding sites of Culicoides pachymerus Lutz in the Magdalena River basin, Colombia.

The breeding sites of Culicoides pachymerus are described for the first time in western Boyacá Province, Colombia, where this species is a public health problem. In addition to being a nuisance due to its enormous density and its high biting rates, C. pachymerus cause dermatological problems in the human population. Analysis of microhabitats by the sugar flotation technique and the use of emergence traps allowed us to recover 155 larvae of Culicoides spp and 65 adults of C. pachymerus from peridomiciliary muddy substrates formed by springs of water and constant rainwater accumulation. These important findings could aid in the design of integrated control measures against this pest.

Behavioral Aspects of Phlebotomine Sand Flies Associated with a Case of Cutaneous Leishmaniasis in Atlántico, Northern Colombia.

After the first autochthonous case of cutaneous leishmaniasis was reported in the Atlántico department in the Caribbean region of Colombia, entomological sampling was conducted in the specific areas where the infection might have occurred. CDC traps were installed inside and outside dwellings in the peri-urban and rural areas of a settlement in the municipality of Luruaco. Sampling was performed during the night with protected human bait, and phlebotomine sand flies were actively sampled from potential diurnal resting sites within dwellings. Ten species of the genus Lutzomyia were identified; Lutzomyia evansi was the dominant species (78%) in the rural and peri-urban areas as well as in the different sampled habitats, followed by Lutzomyia panamensis and Lutzomyia gomezi. There was a 100% household infestation by Lu. evansi, and its indoor mean abundance was 13.3 sand flies/CDC trap/night. The indoor mean abundance of Lu. panamensis and Lu. gomezi was only 0.9 and 0.8 sand flies/CDC trap/night, respectively. Female Lu. evansi were collected with protected human bait, mostly in the peridomestic area, with sustained activity during the night and a slight increase in the activity from 19:00 to 23:00 hours. Of the total sand flies captured in the diurnal resting sites, 73.1% were collected from the walls of bedrooms and corresponded to Lu. evansi, Lutzomyia cayennensis cayennensis, and Lutzomyia trinidadensis. Owing to their vectorial importance, the species on which entomological surveillance should be focused are Lu. evansi, Lu. panamensis, and Lu. gomezi. The biting and resting behavior reported in this study will help guide vector prevention and the control of leishmaniasis within the study area.

Efficacy of Long-lasting Insecticidal Nets With Declining Physical and Chemical Integrity on Aedes aegypti (Diptera: Culicidae).

Fitting long-lasting insecticidal nets (LLIN) as screens on doors/windows has a significant impact on indoor-adult Aedes aegypti (L.), with entomological reductions measured in a previous study being significant for up to 2 yr post-installation, even in the presence of pyrethroid-resistant Aedes populations. To better understand the mode of LLIN protection, bioassays were performed to evaluate the effects of field deployment (0, 6, and 12 mo) and damage type (none, central, lateral, and multiple) on LLIN efficacy. Contact bioassays confirmed that LLIN residual activity (median knockdown time, in minutes, or MKDT) decreased significantly over time: 6.95 (95% confidence interval [CI]: 5.32-8.58) to 9.24 (95% CI: 8.69-9.79) MKDT at 0- and 12-mo age, respectively, using a pyrethroid-susceptible Aedes strain. Tunnel tests (exposing human forearm for 40 min as attractant) showed that deployment time affected negatively Aedes passage inhibition from 54.9% (95% CI: 43.5-66.2) at 0 mo to 35.7% (95% CI: 16.3-55.1) at 12 mo and blood-feeding inhibition from 65.2% (95% CI: 54.2-76.2) to 48.9% (95% CI: 26.4-71.3), respectively; both the passage/blood-feeding inhibition increased by a factor of 1.8-2.9 on LLINs with multiple and central damages compared with nets with lateral damage. Mosquito mortality was 74.6% (95% CI: 65.3-83.9) at 0 mo, 72.3% (95% CI: 64.1-80.5) at 6 mo, and 59% (95% CI: 46.7-71.3) at 12 mo. Despite the LLIN physical integrity could be compromised over time, we demonstrate that the remaining chemical effect after field conditions would still contribute to killing/repelling mosquitoes.

Five new species of Culicoides Latreille described from Colombia, yielding a new species list and country records (Diptera: Ceratopogonidae).

The following five new species of Culicoides from Colombia are described, illustrated and placed to subgenus or species group: Culicoides antioquiensis, Culicoides gabrieli, Culicoides inermis, Culicoides micayensis and Culicoides nigrifemur. C. gabrieli is also known from Peru. When possible, their position in previously published keys is indicated and their features discussed in light of the most recent revisions. A list of 180 Culicoides species known (114) or suspected of being in Colombia (66) is given in a Table. Of these, 12 including the new species are recorded from Colombia for the first time.

[Sand flies (Diptera: Psychodidae) of Guaviare Province, Colombia, with 4 new records for the country]. / Flebótomos (Diptera: Psychodidae) del departamento de guaviare, colombia, con nuevos registros para el país.

INTRODUCTION: Although cases of leishmaniasis have been reported in the province of Guaviare, Colombia, no entomological studies were included to identify the Lutzomyia sand fly vector species in that area. OBJECTIVE: Lutzomyia species were identified from four townships of Guaviare. Probable vectors were named based on those species involved in transmission in other areas. MATERIALS AND METHODS: Sampling was undertaken with CDC light traps suspended at heights between 1.5 m and 9 m. Additional sand flies were collected with Shannon traps and by aspiration of adult flies from daytime resting sites. RESULTS: Sand flies belonging to 37 different species were collected. 35 of them were recorded for the first time in Guaviare Province. Four species were new records for Colombia: Lutzomyia begonae, L. campbelli, L. sericea and L. nematoducta. The most abundant species were L. hirsuta 24.3% (148/610), L. yuilli 15.2% (93/610), L. davisi 10.3% (63/610), followed by L. fartigi, L. carrerai, L. antunesi, L. flaviscutellata and L. olmeca bicolor. CONCLUSION: Seven of these species of have been associated previously with endemic or epidemic transmission of leishmaniasis.

[Preliminary evaluation of the Culicoides biting nuisance (Diptera: Ceratopogonidae) in the province of Boyacá, Colombia]. / Diagnóstico preliminar de la molestia sanitaria causada por Culicoides (Diptera: Ceratopogonidae) en el departamento de Boyacá, Colombia.

INTRODUCTION: Inhabitants in the western border of Boyacá province have reported high nuisance levels and dermatologic problems caused by the intensely irritating bites of the very small flies of the genus Culicoides. OBJECTIVE: A survey was carried out to locate the affected area, identify the anthropophylic Culicoides species and estimate its abundance in Boyacá. MATERIALS AND METHODS: Nuisance reports and clinical records of dermatologic cases associated with Culicoides bites were requested from health authorities in counties where nuisance reports had been received or which had geographical features apparently favorable for Culicoides infestations. An outdoors entomological survey using human landing catches was undertaken in areas reporting a pest problem. RESULTS: Culicoides infestations were confirmed as a serious nuisance problem in the rural areas of nine counties located in the western foothills of the Eastern Range of the Colombian Andes. Although available epidemiological records were fragmented, it was established that in six counties 11.4% of the dermatitis cases (total=2,472 cases) reported between 2003 and 2005 were attributed to the Culicoides bites. The entomological survey identified Culicoides pachymerus as the dominant species, 99.3% of 3,389 caught females. Biting rates in the most intensely affected areas reach a geometric mean of 52 females/person per 5 minutes. Multivariate analysis indicated that abundance of C. pachymerus had a negative relationship with altitude. CONCLUSIONS: Based on its dominance and high biting rates, C. pachymerus is probably the species responsible for the high nuisance levels caused by Culicoides bites and the associated dermatological pathology, within the study area.

[Presence of infected vectors of Leishmania (V.) panamensis within dwellings in two endemic foci in the foothill of the middle Magdalena valley, western Boyacá, Colombia]. / Presencia en el peridomicilio de vectores infectados con Leishmania (Viannia) panamensis en dos focos endémicos en el occidente de Boyacá, piedemonte del valle del Magdalena medio, Colombia.

INTRODUCTION: Case records of leishmaniasis of the years 1997 to 2003 of the department of Boyaca showed that since the year 2000 the department experienced an unusual rise in the incidence of cutaneous leishmaniasis that might correspond to an epidemic outbreak in the western region of the department. Age and gender distribution of cases supported a domestic transmission. OBJECTIVE: This research was designed to identify the vectors of cutaneous leishmaniasis in the municipalities of Otanche and Pauna through their presence in dwellings and their natural infection with the same species of Leishmania isolated from patients. MATERIAL AND METHODS: Sampling of sand flies was done with CDC traps in and around dwellings. Samples from patients and pooled females of the most abundant species of Lutzomyia were used to identify the parasite by PCR. Monoclonal antibody typing was also used to confirm the identification of the parasite in samples from patients. RESULTS: In both municipalities L. trapidoi was the most abundant anthropophilic species of Lutzomyia indoors and around dwellings. L. hartmanni and L. yuilli were also abundant in Otanche, and L. gomezi and L. panamensis in Pauna. Leishmania (V) panamensis was identified both in patients and in the sand flies: L. yuilli, L. gomezi and L. panamensis.. CONCLUSION: Our findings prove the presence of infected vectors of Leishmania panamensis within dwellings in the towns of Otanche and Pauna in Western Boyacá. Since L. trapidoi was the most abundant species, it may be considered as the principal vector of Leishmania (V.) panamensis. The evidence of transmission within human dwellings warrants vector control at least in this environment.

[Validation of PCR as a tool for the detection of Leishmania (Vianna) spp. parasites in the Lutzomyia (Diptera: Psychodidae) vector]. / Validación de la PCR en la detección de parásitos de Leishmania (Viannia) spp. en Lutzomyia (Diptera: Psychodidae) como herramienta en la definición de especies vectores.

INTRODUCTION: The applicability of the polymerase chain reaction (PCR) was evaluated for the detection and identification of parasites in sand fly vectors and thereby precluding the necessity of dissecting them. DNA was extracted from individual, laboratory infected sand flies, and subjected to PCR amplification using specific B1 and B2 primers for parasites of the Leishmania (Viannia) subgenus. METHODOLOGY: The sensitivity and specificity of the PCR primers were defined by means of serial dilutions of a Leishmania culture. Pooled samples of 1 to 5 sandflies were examined in association with the parasite dilutions to determine the point at which sensitivity became reduced. Experimentally infected sand flies were used to compare the sensitivity of the PCR with sand fly dissection and searching for flagellated parasites by microscopic examination. RESULTS: As few as a single parasite was detected, and the sensitivity remained unaltered up to 3 female sand flies per pool. Detection rates were 33% for the traditional technique and 33.3% for PCR. The B1 and B2 primers were confirmed as specific for Leishmania (Viannia) parasites. CONCLUSION: The demonstrably high sensitivity and specificity of PCR warrant the use of PCR in assessing natural infection rates of Leishmania (Viannia) in field populations of sand fly vectors.

Efficacy of factory-treated and dip-it-yourself long lasting insecticide-treated bednets against cutaneous leishmaniasis vectors in the sub-Andean region of Colombia: results after two years of use

BACKGROUND Long lasting insecticide-treated nets (LLINs) may be effective for vector control of cutaneous leishmaniasis (CL). Their efficacy, however, has not been sufficiently evaluated. OBJECTIVE To evaluate the large-scale efficacy of LLINs on Lutzomyia longiflocosa entomological parameters up to two years post-intervention in the sub-Andean region of Colombia. METHODS A matched-triplet cluster-randomised study of 21 rural settlements, matched by pre-intervention L. longiflocosa indoor density was used to compare three interventions: dip it yourself (DIY) lambda-cyhalothrin LLIN, deltamethrin LLIN, and untreated nets (control). Sand fly indoor density, feeding success, and parity were recorded using CDC light trap collections at 1, 6, 12, and 24 months post-intervention. FINDINGS Both LLINs reduced significantly (74-76%) the indoor density and the proportion of fully engorged sand flies up to two years post-intervention without differences between them. Residual lethal effects of both LLINs and the use of all nets remained high throughout the two-year evaluation period. CONCLUSIONS Both LLINs demonstrated high efficacy against L. longiflocosa indoors. Therefore, the deployment of these LLINs could have a significant impact on the reduction of CL transmission in the sub-Andean region. The DIY lambda-cyhalothrin kit may be used to convert untreated nets to LLINs increasing coverage.

Estimating carrying capacity in a newly colonized sand fly Lutzomyia serrana (Diptera: Psychodidae).

The phlebotomine sand fly Lutzomyia serrana (Damasceno & Arouck) was mass-reared tinder conditions of varying densities in an effort to improve colony production efficiency. To do this, the experimental carrying capacity of a standard rearing chamber was determined, i.e., the optimum population size in relation to density (individuals per unit of space). Rearing chambers of 100 cm3 were populated with 1-50 L. serrana engorged females and an equal number of males. Laboratory conditions were maintained at 23-26 degrees C and 85-95% RH. The following parameters were recorded for each experimental chamber (three replicates): (1) female mortality without oviposition, (2) number of eggs oviposited and (3) number of adults emerging from the egg cohort. Female mortality began to increase substantially in the 26-female chamber, from 5.7% to 15% and finally reaching 60.2% in the 46-50 female chambers. In the chambers containing 1-20 females, egg number and realized adult progeny increased linearly to reach an asymptote. In the 20-50 female chambers, the number of eggs ranged from 420 to 699, and adult production from 306 to 432. The optimum carrying capacity for the 100-cm3 chambers was 22 +/- 2 females. Beyond this number, auto-regulation was initiated, i.e., female mortality without oviposition increased as the number of females per chamber increased. Total number of eggs and adult production was similar in all chambers containing 20-50 females. In conclusion, for optimizing production of mass reared sand flies, determination of the carrying capacity is essential to optimize use of insectary resources, to avoid loss of valuable potentially ovipositing females, and to increase overall production efficiency.