MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 95(2) Mar/Apr 2000
PAGES: 251-257 DOI: Full paper
Aspects Related to Productivity for Four Generations of a Lutzomyia longipalpis Laboratory Colony

José Francisco Luitgards-Moura +, Eloy Guillermo Castellón BermúdezI, Maria Goreti Rosa-FreitasII

Núcleo Avançado de Vetores, Convênio Fiocruz-UFRR, CCBS Medicina, BR 174 s/nº, 69310-270 Boa Vista, RR, Brasil
IInstituto Nacional de Pesquisas da Amazônia, Caixa Postal 478, Manaus, AM, Brasil
IILaboratório de Sistemática Bioquímica, Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brasil

Abstract

A closed colony of Lutzomyia longipalpis was established with specimens collected in the Raposa _ Serra do Sol indian reservoir, one of the main foci of visceral leishmaniasis in the State of Roraima, Brazil. Biological observations were made on four generations of a L. longipalpis colony with emphasis on productivity. Aspects studied were the number of laid and retained eggs, and the number of adults (male and female) per generation. During the four generations the percentage of engorged females that laid eggs varied from 64.2% (third generation-F3) to 90.3% (second generation-F2). The mean number of eggs laid per female varied from 23.6 (F3) to 39.9 (first generation-F1). The maximum number of eggs laid per female varied from 84 (F3) to 124 (F1). The mean number of retained eggs per female was 12.7 (parental generation-P and F1) to 22.1 (F2). The number of females exceeded the number of males in all generations. However, significant difference for male/female ratio was found only for F3. Fecundity rates were between 42.1 (F3) and 58.3 (F2). From a total of 439 blood-fed females, 355 females laid 12,257 eggs that yield 5,354 adults (2,525 males and 2,829 females) in four generations. F2 presented maximum productivity and fecundity rates.

Lutzomyia longipalpis is the main visceral leishmaniasis vector in Brazil (Deane 1956). Because of the need of adults for natural insecticide bioassays (to be shown elsewhere) a L. longipalpis colony was initiated. Colonies of L. longipalpis have been successfully established before (Sherlock & Sherlock 1959, Killick-Kendrick et al. 1977, Ready 1978,1979, Modi & Tesh 1983, Buescher et al. 1984). L. longipalpis populations from Central and South America show a great polymorphism in behavior, morphology and biochemical characters (Ward et al. 1983, Hamilton et al. 1996, Dujardin et al. 1997, Mukhopadhyay et al. 1998a, b, Dias et al. 1998, Yin et al. 1999). These results either lead L. longipalpis differences to be suspected as due to a complex of cryptic species (Lanzaro et al. 1993, Warburg et al. 1994, Hamilton et al. 1996, Dujardin et al. 1997, Yin et al. 1999) or within range of intrapopulational variation (Azevedo 1998, Mutebi et al. 1999). L. longipalpis specimens from Roraima population had not been studied before regarding its productivity in captivity. Roraima presents ecological conditions diverse from the Amazon Region. Savanna accounts for 25% of the Roraima vegetation (75% being tropical rain forest) and rainfall regimen is opposite to the rest of the Amazon. Specimens were collected in a leishmaniasis focus in Maloca do Napoleão (Raposa - Serra do Sol indian reservoir) where the dry period extends up to eight months per year. Methods used in previous colony establishment, mainly those of Killick-Kendrick et al. (1977) and Ready (1978), were used as guidelines for colonizing L. longipalpis specimens collected at Maloca do Napoleão, Roraima, Brazil. Aspects related to productivity were studied and compared to previous findings. These aspects included mean and maximum number of laid and retained eggs per female and per generation and, sex ratio. Productivity and fecundity were calculated.

 

MATERIALS AND METHODS

Seventy nine L. longipalpis females were collected in CDC light traps set from 18:00 to 6:00 h during four days in November 1996. These traps were placed in the peridomicile of a house and inside a chicken roost, located in the vicinity of a dam, in a place known as Maloca do Napoleão. This endemic visceral leishmaniasis area is located in the Normandia municipality (03°52'52''N, 59°37'22''W, 100 m), Roraima, Brazil. The region is formed by savanna, with a tropical humid climate and maximum rainfall of 1,600 mm in June (Ministério das Minas e Energia 1975). Local population is mainly formed by Macuxi indians. CDC trapped phlebotomines were transferred to nylon cages (22 cm3) and blood-fed on a hamster. Engorged females were transported in screw-cap plastic pots (Nalgene 11 cm x 7 cm height, 500 ml capacity), modified. These modifications consisted in cutting off two circles of 9.5 cm diameter from the cap and the bottom. The cap when closed hold a 20 cm2 piece of nylon gauze. The bottom was replaced by pouring freshly prepared plaster up to 2 cm height. After the bottom was dried, plaster was poured on the sides of the pot. Pots containing collected phlebotomines were closed in plastic bags with cotton wool and ice, inside a polystyrene box until arrival in the insectary in Manaus, State of Amazonas. A slice of apple was placed on the top of the nylon gauze. Phlebotomine females were identified according to taxonomic keys (Ryan 1986). Rearing procedures followed Killick-Kendrick (1987) with a few modifications. The modifications introduced were the substitution of filter paper for sterilized sand inside snap-cap plastic boxes for maintenance of humidity level and the change of sucrose solution for apple slices as additional food source for the adults. Females were blood-fed four days after emergence in anesthetized hamsters. Thionembutal given intraperitoneally was the anesthetic used. Dose varied from 0.4 mg/10 g hamster body weight to 15 mg/10 g body weight, as tolerance increased. Engorged females were individually transferred to 30 ml glass vials with a piece of wet filter paper to lay eggs. Labels containing dates of blood meal, oviposition and death were placed in those vials. Colony was maintained in the facilities of the Leishmaniasis Laboratory (Coordenação de Pesquisas em Ciências da Saúde, Instituto Nacional de Pesquisas da Amazônia, INPA, Manaus). Insectary temperature and humidity were kept at 25-28ºC and 75-80%, respectively. Data presented refers to November 1996-October 1997, when the colony reached the fourth generation (F4). After blood meal, slices of apple were placed on the nylon gauze and changed every day. Egg production was verified daily. Eggs were counted and separated in plaster lined plastic pots (Nalgene 11 cm x 7 cm height, 500 ml capacity) to develop to larvae and pupae. Larvae of all stages were daily fed with a 1:1 mixture of ground and sieved rabbit food and rabbit feces (Modi & Tesh 1983) kept at -40°C. Pots had their plaster lining changed after one week without adult emergency observation. The plaster change aimed to avoid fungal and mite contamination. Pots were kept in closed 20 x 20 cm snap-cap plastic boxes with sterilized sand to maintain the humidity, and avoid mite infestation. After oviposition, females were dissected for observation of the number of eggs retained in their ovaries. The number of male and female adults emerged were counted daily and transferred to nylon cages. These cages were tightly closed in plastic bags with a wet cotton-wool ball inside. A dark cloth was placed around plastic bags to maintain adults in dark conditions. Adult emergence was checked at 7:00 h and 19:00 h daily. Fecundity rate for each generation was determined by dividing the total number of eggs (laid plus retained) by the number of females. Productivity was calculated by dividing the number of adults (males and females) by the number of eggs laid (x 100) for each generation. Statistical analysis used Statigraphics v. 5.01 and Systat v. 5.01 software programs.

 

RESULTS

Males caught in Normandia, Roraima, presented a one-spot phenotype, i.e., tergite IV with paired pale patches of pheromone glands (Ward et al. 1983). Adults copulated 1 to 2 days after emergence irrespective of females being blood-fed. Oviposition occurred from 6 to 13 days after females were blood-fed on a hamster (data not shown). Females readily fed on hamsters in either dark or light conditions. The percentage of blood-fed females that laid eggs varied from 64.2% (in the third generation-F3) to 90.3% (second generation-F2, Table I). The great majority of females died after 24 h after oviposition (data not shown). No females were blood-fed a second time. Fungicides have been used in sand flies colonies for the control of fungus contamination (Hertig & Johnson 1961, Schmidt 1964). However, mite and fungal contaminations were reduced by a few measures, as changing plaster every time larvae reached adult stage, by maintaining larval food in freezer (-40°C) and by flaming rearing tools. Even though not quantified, it was observed that males emerged 2-3 days before females. Adult emergence, also not quantified, occurred mainly at the morning checking time (7:00 h). The mean number of eggs laid per female was 23.6 (± 26.3) for F3 to 39.9 (± 27.5) for F1 (Table I). The number of eggs laid by females in the parental generation-P is unknown, as previous oviposition may have occurred prior to collection. For the same reason, the total number of eggs, the number of males and females yield, fecundity and productivity rates could not be obtained for P. The maximum number of eggs laid per female varied from 84 in the F3 to 124 in the first generation-F1 (Table I). The number of eggs laid per generation was in the range of 2,236 (F3) to 3,919 (F1, Table I). The mean number of retained eggs varied between 12.7 (± 14.9-P and ± 18.9-F1) and 22.1 (± 24.0-F2,Table II). The maximum number of retained eggs per female was 62 for P to 78 in the F2 (Table II). The number of retained eggs per generation varied from 950 (fourth generation-F4) to 2,059 (F2, Table II). Total number of eggs produced (laid plus retained) per generation varied from 3,687 (F4) to 5,424 (F2, Table III). Kruskall-Wallis and Dunn statistical tests showed that there was a significant difference (p<0.001) for the mean number of laid eggs, for the mean number of retained eggs and, for total eggs produced among the four generations. In relation to fecundity, rates were from 42.1 (F3) to 58.3 (F2, Table IV). When accounted together the four generations exhibited a fecundity rate of 41.6 (Table IV). A total of 79 parental generation females yield in captivity 395 males and 432 females in the F1 generation (0.91 male/ female ratio, Table V). Male/female ratio varied from 0.79 (F3) to 0.96 (F4, Table V). Statistical tests showed a significant difference in the male/female ratio only for F3 (p<0.001, Table V). F2 produced the maximum number of males (1,105) and females (1,179) from 3,365 eggs laid by 84 females, presenting the highest productivity (67.9%, Table V). F1 showed the poorest productivity 21.1%, with 395 males and 432 females emerged from 3,919 eggs laid by 86 females (Table V). For a total of 12,257 eggs laid from 355 females in four generations of L. longipalpis, 5,354 adults emerged (2,525 males and 2,829 females) with an overall productivity of 43.7% (Table V). A summary of the results is given in Table VI. A total of 18, 283 eggs (12,257 laid eggs and 7,037 retained eggs) was obtained during four generations of L. longipalpis in captivity (Table VI).

 

DISCUSSION

L. longipalpis has long been recognized as the main visceral leishmaniasis vector in Brazil (Deane 1956). A L. longipalpis colony was initiated for natural insecticide bioassays (to be shown elsewhere) with plants and insects collected in Roraima, a State in the Brazilian Amazon. Methods used for previous L. longipalpis colonies were used aiming to achieve maximum productivity (Killick-Kendrick et al. 1977). L. longipalpis specimens from Roraima population have not been studied before regarding its productivity in captivity. Roraima presents ecological conditions diverse from the Amazon Region, regarding vegetation (25% savanna and 75% tropical rain forest) and rain fall regimen (opposite to the rest of the Amazon) that could account for differences in the local population. Specimens were collected in a leishmaniasis focus in Maloca do Napoleão (Raposa -Serra do Sol indian reservoir). The aspects studied were the mean and maximum number of laid and retained eggs per female and per generation and, sex ratio for four generations of L. longipalpis in captivity. Based on these results, productivity and fecundity were calculated.

Even though morphological analysis was not aimed, it could be observed that males from this population were the one-spot phenotype (Ward et al. 1983). Variability in tergal spot morphology is believed to be a polymorphic character not related to genetic isolation (Mukhopadhyay et al. 1998a).

Time between blood meal and oviposition varied from 6-13 days (data not shown). Previous works report a variation from 2 to 7 days (Sherlock & Sherlock 1972) and 4 to 9 days (Killick-Kendrick et al. 1977).

Males emerged two days before females (data not shown). After this period, male/female ratio was bigger for females (Table VI). Early male emergence has been observed for many insects. It has been long known the male genitalia of some insects undergoes a 180° rotation after emergence (Clements 1992 for review on Culicidae). The 180° male genitalia rotation and late female emergence (2-3 days after male emergence) seem to be linked. Genitalia rotation is necessary for male to become sexually mature. When females emerge, males are ready for copulating. Time for pheromone production may also account for time emergence discrepancy between males and females. As many as four days are necessary for pheromone production to be maximum in males and a threshold level might be necessary to attract females (Hamilton et al. 1994). Also pheromone production may help sclerotization of the aedeagus.

Combined extract of rabbit food, rabbit feces and larval rearing media showed to have a synergetic effect on sandfly egg laying (Elnaiem & Ward 1992). Nonetheless we opted for not only cleaning the pots but also change the plaster as mite and fungus were a problem for colony establishment.

Adults were checked for emergence at 7:00 h and 19:00 h, daily. Adults were more abundant at the 7:00 h checking time, indicating a preference for night/early morning emergence. The cool and moist time near dawn is long known to be the time of choice for emergence for most insects (Pittendrigh 1957). However, an hourly checking should have been done to accurately determine that night was the preferential emergence time.

The difference for sex ratio emergence in the four generations with predominance of females may be due to fluctuations on genes that alter sexual proportion. These fluctuations might be natural oscillations or the beginning of a gene fixation (Cazemajor et al. 1997, Owusu-Daaku et al. 1997 for Aedes aegypti). Killick-Kendrick et al. (1977) observed a ratio of males to females of 1.2:1.0 for L. longipalpis (Lapinha strain).

The fact that almost all females died within 24h of oviposition has been observed before (Killick-Kendrick et al. 1977, Buescher et al. 1984, Elnaiem & Ward 1992).

Souza et al. (1995) state that optimal conditions of laboratory maintenance relate to both sugar and blood feeding. In fact, Brazil and Ward (1989) suggest that L. longipalpis females lay more eggs when they receive sucrose. Size and composition of blood meal also affect egg laying (Ready 1979). Females were fed to completion in hamsters and slices of apple were their source of sugar.

Females laid an average of 23.6 (F3) to 39.9 (F1) eggs per oviposition (Table VI). This is in accordance to previous findings of 30.1 eggs per oviposition (mean, Sherlock & Sherlock 1959). The mean and maximum number of eggs laid per female, males and females produced, fecundity and productivity in the parental generation could not be obtained, as parental females age and parity status in the field prior to collection are unknown.

Fecundity and productivity are aspects that aim to indicate the success of a given generation. Nonetheless, this meaning should be used with parsimony. Fecundity rate is related to egg production irrespective of oviposition. A generation is said fecund even if retains eggs produced. Productivity relates to the number of adults emerged from a certain number of eggs laid. Productivity is high even when a small number of laid eggs originate the most amount of adults. For productivity the number of retained eggs is irrelevant.

In spite of having the highest number of retained eggs per female (22.1) and per generation (2,059), F2 had the highest number of eggs produced (5,424 from 84 blood-fed egg-laying females) and the highest adult emergence (2,284 adults out from the 3,365 eggs laid). As a result of these features F2 was the most fecund (58.3) and productive generation (67.9%, Table VI).

The lowest productive generation F1 retained 1,252 eggs (the third in the four generations), had the highest number of laid eggs per female (39.9) and per generation (3,919) but only 827 emerging adults. Therefore F1 ranked last for productivity (21.1, Table VI), although was the second highest for fecundity (52.8, F2 was the highest with 58.3, Table VI).

Generation performance also relates to the individual level. A total of 90.3% of F2 females were egg-laying females (the highest percentage, Table VI). F2 also presented the second highest number of eggs laid per female (94 opposed to 124 for F1, Table VI) and in spite of showing the highest number of eggs retained per female (78, Table VI) attained the highest fecundity (58.3) and productivity (67.9%, Table VI). Even though F1 had the highest number of eggs laid per female (124) and the second least number of retained eggs (70 opposed to 50 for F4, Table VI), had the lowest numbers of emerged adults and the lowest productivity (21.1, Table VI).

A successful generation can be assessed by fecundity and productivity that bring in their definitions the interconnected, interrelated variables (genome of parental founder females, nutrients availability throughout the life cycle, developmental environment) behind a high percentage of blood-fed females able to lay eggs, a large number of laid eggs (irrespective of the number of retained) and a high number of adults emerged.

 

ACKNOWLEDGEMENTS

To Rui Alves de Freitas for helping in the taxonomy, Toby Vincent Barrett, Roberto Dualibi Naiff and Raul Guerra de Queiroz for their advisory and Roberto Farias and Silvia Cassia Brandão Justiniano for technical support, Alexandre Afrânio Peixoto for fruitful discussion on insect behavior and two anonymous referees for their invaluable contributions.

 

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Cazemajor M, Landré C, Montchamp-Moreau C 1997. The sex-ratio trait in Drosophila simulans: genetic analysis of distortion and suppression. Genetics 147: 635-642.

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