Cobalt chloride induces metaphase when topically applied to larvae and pupae of the stingless bee Melipona scutellaris (Hymenoptera, Apidae, Meliponini).

In order to optimize preparations of bee metaphases, we tested cobalt chloride, which has been used as a metaphase inducer in other organisms, such as hamsters and fish. Four microliters of 65 mM cobalt chloride aqueous solution was topically applied to larval and pupal stages of the stingless bee Melipona scutellaris. The cerebral ganglion was removed after treatment and prepared for cytogenetic analysis. Identically manipulated untreated individuals were used as controls. The number of metaphases was increased 3-fold in treated individuals compared to controls. The micronucleus test showed no mutagenic effects of cobalt chloride on M. scutellaris cells. We concluded that cobalt chloride is a metaphase-inducing agent in M. scutellaris, thus being useful for cytogenetic analyses.

Number of malpighian tubules in larvae and adults of stingless bees (Hymenoptera: Apidae) from Amazonia.

The number of Malpighian tubules in larvae and adults of bees is variable. Larvae of Apis mellifera L. have four Malpighian tubules, while adults have 100 tubules. In stingless bees, this number varies from four to eight. The objectives of this study were to provide characteristics of the Malpighian tubules as well as to quantify their number in larvae and adults of six species of Meliponinae, Melipona seminigra merrillae Cockerell, Melipona compressipes manaosensis Schwarz, Melipona rufiventris Lepeletier, Scaptotrigona Moure, Frieseomelitta Ihering, and Trigona williana Friese. Malpighian tubules were dissected from larvae and adults, measured, quantified, and maintained in microtubes with Dietrich's solution. The numbers of Malpighian tubules were constant only for larvae of M. rufiventris (four and eight) and Scaptotrigona sp. (four). The most frequent number of tubules in the Melipona group was seven and eight in larvae, and 70 and 90 in adults. In the Trigona group were four and 20 to 40, for larvae and adults, respectively. The results showed differences in the number of Malpighian tubules among the species analyzed and also between the larvae and adults of the same species. Despite the variation observed, species of the group Melipona always have a larger number and longer Malpighian tubules in both larvae and adults as compared to the Trigona group, which may indicate an evolutionary trend of differentiation between these groups.

Does beekeeping reduce genetic variability in Melipona scutellaris (Apidae, Meliponini)?

Many factors have contributed to reductions in wild populations of stingless bees, such as: deforestation, displacement and destruction of nests by honey gatherers, as well as use of insecticides and other agrochemicals. All of these can potentially affect the populational structure of native species. We analyzed genetic variability and populational structure of Melipona scutellaris, based on five microsatellite loci, using heterologous primers of M. bicolor. Samples were taken from 43 meliponaries distributed among 30 sites of four northeastern states of Brazil (Pernambuco, Alagoas, Sergipe, and Bahia). Thirty-one alleles were found to be well distributed among the populations, with sizes ranging from 85 to 146 bp. In general, there was a variable distribution and frequency of alleles among populations, with either exclusive and/or fixed alleles at some sites. The population of Pernambuco was the most polymorphic, followed by Bahia, Alagoas and Sergipe. The heterozygosity was Ho = 0.36 on average, much lower than what has been reported for M. bicolor (Ho = 0.65). Most populations were not under Hardy-Weinberg equilibrium. We found a higher variation within rather than among populations, indicating no genetic structuring in those bees maintained in meliponaries. This apparent homogenization may be due to intense beekeeping activity, including exchange of genetic material among beekeepers. Based on our findings, we recommend more studies of meliponaries and of wild populations in order to help orient management and conservation of these native pollinators.

A mixed colony of Scaptotrigona depilis and Nannotrigona testaceicornis (Hymenoptera, Apidae, Meliponina).

We describe a case of a spontaneously established mixed colony of two species of stingless bees. The host colony of Scaptotrigona depilis, an aggressive bee that forms large colonies, was invaded by workers of Nannotrigona testaceicornis, a smaller bee that forms small colonies. The host colony and the invading species colony were maintained in next boxes about 1.5 m apart. The N. testaceicornis colony had been recently divided. Observations were made daily for 10 min, and every two weeks the colony was opened for observations within the nest. Initially the host colony bees repulsed the invading species, but as their numbers built up, they were no longer able to defend the entrance. An estimated 60-90 N. testaceicornis workers lived integrated into the colony of S. depilis for 58 days. During this period, they reconstructed and maintained the entrance tube, changing it to an entrance typical of N. testaceicornis. They also collected food and building material for the host colony. Nannotrigona testaceicornis tolerated transit of S. depilis through the entrance, but did not allow the host species to remain within the tube, though the attacks never resulted in bee mortality. Aggression was limited to biting the wings; when the bees fell to the ground they immediately separated and flew back. There have been very few reports of spontaneously occurring mixed stingless bee colonies. It is difficult to determine what caused the association that we found; probably workers of N. testaceicornis got lost when we split their colony, and then they invaded the colony of S. depilis.

Infestation by Pyemotes tritici (Acari, Pyemotidae) causes death of stingless bee colonies (Hymenoptera: Meliponina).

We report the infestation of stingless bee nests by the mite Pyemotes tritici, which killed four colonies of Tetragonisca angustula and one colony of Frieseomelitta varia in Brazil. The first infected colony, a colony of T. angustula, came from an area between Uberlândia and Araguari, Minas Gerais. The transfer of the mites to the other colonies occurred through the transfer of infected combs and subsequent manipulations. Other colonies in the same meliponary, which had not been manipulated, were not infected. The infestation was terminated by isolating the dead colonies from the meliponary.

Climatic and anthropic influence on size and fluctuating asymmetry of Euglossine bees (Hymenoptera, Apidae) in a semideciduous seasonal forest reserve.

We examined the influence of climate and man on size and fluctuating asymmetry in two species of Euglossine bees collected from a semideciduous forest reserve. Sixty males of each species were collected; four measurements were made of their wings to obtain a multivariable size index and a fluctuating asymmetry index. No significant differences in the size of Eulaema nigrita Lepeletier were found between the areas and seasons. Larger males of Euglossa pleosticta Dressler were collected during the hot and wet season; however, male size did not vary with location. Higher rainfall and a consequent increase in food availability could have influenced the increase in size of E. pleosticta. Bees collected during the hot and wet season at the forest border were more asymmetric than bees collected during the cold and dry season; the latter were found inside the forest. This indicates that climate and anthropic interferences influence the stability of development of E. pleosticta. Consequently, this species could be used as a bioindicator of stress. Apparently, E. nigrita is more resistant to environmental interference.

Effects of juvenile hormones I, II and III, in single and fractionated dosage in Melipona bees.

Applications of Juvenile Hormones JH I, II and III in single or fractionated doses to larvae of Melipona compressipes, Melipona quadrifasciata, Melipona rufiventris and Melipona scutellaris at the L3 and beginning of cocoon-spinning phase (pre-defecating larvae - LPD) activated feminizing genes, inducing differentiation of female larvae into queens. The technique of fractionated treatment proved to be highly efficient in Melipona species for producing of queens. It is difficult to obtain 100% queens with single dosage of JH in some species which implies in a mechanism of JH degradation by specific esterases. One hundred per cent of queen production occurs only when an adequate amount of Juvenile Hormone is administered within the period that is critical for caste determination. The threshold dosage of JH I for Melipona compressipes (below which it is not possible to obtain 100% queens) was 0.1mu JH I 4 applications of 0.25mu g each. For Melipona quadrifaciata the threshold dose for obtaining the highest proportion of queens was 0.2mu g JH I in 4 applications of 0.05mu g each. For Melipona scutellaris 0.025mu g JH I/mj 1 in single dosage induced 100% of queens. No 100% production of queens in Melipona Rufiventris treated larvae were obtained; the highest proportion was 86% of queens with 0.2mg g JH I/mu 1. In order to obtain expression of the feminizing genes in Melipona species, JH I was the most efficient followed by JH III. Each species respond differentialy to JH dosage, and this dosage must be tested before use.

Probable inbreeding effect on male homosexuality.

O coeficiente de endocruzamento (F) de uma amostra de homossexuais masculinos (0.00781) mostrou-se cerca de 18 vezes mais elevado que o estimado para a populacao geral. Este fato e a concentracao familiar verificada sugerem que, apesar de se tratar de um grupo etiologicamente heterogeneo, a homossexualidade masculina parece ter um pequeno componente genetico

Estimation of the number of sex alleles and queen matings from diploid male frequencies in a population of Apis mellifera.

The distribution of diploid males in a population of Apis mellifera was obtained by direct examination of the sexual phenotypes of the larvae. Using these data, estimates are derived for the number of sex alleles and the number or matings undergone by the queen. The number of sex alleles is estimated to be 18.9. The estimate is larger than previous ones, which have ranged between 10 and 12. However, the increase in the number of sex alleles can be explained by the large effective population number for our data. The best estimator of the number of matings by a queen is a maximum likelihood type that assumes a prior distribution on the number of matings. For the data presented here, this estimate is 17.3. This estimate is compared to others in the literature obtained by different approaches.

Teratogenic and toxicological examination of 2,4,5-trichlorphenoxyacetic acid in developing chick embryos.

Practical grade 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), dissolved in dimethylsulfoxide (DMSO), was injected into the air space of fertilized chicken eggs prior to incubation. Doses of 2,4,5-T administered were 12.5, 25, 50, 75, 100, and 125 mg/kg to determine herbicide toxicity on the first day of incubation. A similar group was studied on day 5 of incubation with doses of 2,4,5-T at 50, 75, 100 and 250 mg/kg. LD50 was estimated to be 62 mg/kg on day zero and 68 mg/kg on day 5. Additionally, embryos were exposed to 2,4,5-T at 50 mg/kg on day zero of gestation and sacrificed after 48 h of incubation. Serial sections were examined for teratological and developmental anomalies. None were found.

Sex determination in bees. IV. Genetic control of juvenile hormone production in Melipona quadrifasciata (Apidae).

Cell number and volume of corpora allata was determined for 8 phases of development, the first prepupal stage to adults 30 days old, in the social Apidae Melipona quadrifasciata. In the second prepupal stage a strong correlation was found between cell number and body weight (r = 0.651), and cell number and corpora allata volume in prepupal stage (r = 0.535), which indicates that juvenile hormone has a definite role in caste determination in Melipona. The distribution of the volume of corpus allatum suggest a 3:1 segregation between bees with high volume of corpora allata against low and medium volume. This implies that genes kappa a and kappa b code for an enzyme that directly participates in juvenile hormone production. It was also concluded that the number of cells in the second prepupal stage is more important than the weight of the prepupa for caste determination. A scheme summarizing the genic control of sex and caste determination in Melipona bees in the prepupal phase is given.

Genetic component in learning ability in bees.

Twenty-five bees, five from each of five hives, were trained to collect food at a table. When the bee reached the table, time was recorded for 12 visits. Then a blue and yellow pan was substituted for the original metal pan, and time and correct responses were recorded for 30 trips (discrimination phase). Finally, food was taken from the pan and extinction was recorded as incorrect responses for 20 visits. Variance analysis was carried out, and genetic variance was undetected for discrimination, but was detected for extinction. It is concluded that learning is very important for bees, so that any impairment in such ability affects colony survival.

Population genetic studies in bees (APIDAE, Hymenoptera). I. Genetic load.

Three populations of Apis mellifera each predominantly of a different subspecies (mellifera, ligustica and adansonii) and 7 species of stingless bees (Meliponinae, Apidae) were manipulated for applying the MORTON, CROW & MULLER's methodology in order to estimate the lethal equivalents (B) of each population. A total of 249 queens were used, 27 being meliponids and 222 Apis mellifera. The populations of Apis have a B that does not differ significantly when they are compared to each other (1.29, 1.36, 1.32) and the balanced average equals 1.33. When the x-alleles are not considered, this balanced value is 0.262. The figures for B in the seven species of stingless bees ranged between 0.104 and 0.159 with balanced average of 0.132. The main reason for this smaller load in meliponids may rest in their effective population numbers, which are smaller than those of Apis. The average mortality for diploid females (0.141) and for haploid males (0.163) allows the estimation of the total elimination (sigma E = 0.073). Since for haplo-diploid systems the total mutation rate is sigma mu = 2 sigma E divided by 3 the figure 0.048 is obtained. Since about 15% of the genes in Apis mellifera are sex limited, this value of sigma mu should be added of 0.0072 (that is 0.15 X 0.048) and then, the total mutation rate becomes 0.055. Using a quite different method, the one by MORTON, CROW & MULLER, the figure 0.076 was obtained. If a mutation rate of 10(-5) is assumed, the number of genes in Apis mellifera that can make a contribution to the genetic load would vary between 5,500 and 7,600.