The Western honey bee (Apis mellifera) is the most beneficial insect to humans. In the United States, the value of agricultural output increased by pollination amounts to US$19 billion each year (Morse and Calderone, 2001). Studying the social behavior of bees can enable humans to manage bees more scientifically and serve agricultural production. Studying the biology of bee pests and diseases and their control can lead to healthier bee colonies. Therefore, research on bee behavior and pest and disease control has always been a hot topic in basic and applied research internationally. In the past ten years, Huang Zhiyong has completed a number of national research projects as the main researcher or project leader. The results achieved are summarized below:
1. Establishing a model of "social inhibition" and identifying a fourth initiating pheromone in animals.
The main basis of bee social behavior is "age division of labor", that is, worker bees of different ages do different jobs in the colony. Years of work experience have taught Huang Zhiyong that there is a big difference in the physiological state of office bees, represented by nurse bees, and field bees, represented by collector bees, and this difference is mostly affected by juvenile hormone (JH) or controlled. However, what kind of information in the nest controls worker bee juvenile hormone has long been a mystery. Borrowing methods commonly used in developmental biology, worker bees were raised individually in isolation. It was found that the juvenile hormone and behavioral development of worker bees developed in advance, and they became "collector bees" (with high JH concentration and display of collecting behavior in the colony) within seven days. ). They also used the method of "transplanting" collector bees and found that "premature" collector bees can be produced in "swarms of the same age" (a colony composed of newly emerged worker bees and queen bees, without collector bees) in 5-7 days. , can be suppressed by transplanted collector bees. Based on these experimental results, Huang and Robinson (1992) published a model in PNAS, arguing that the behavioral development of worker bees from nurse bees to foragers is mainly regulated by the ratio of foragers to nurse bees in the colony. There is a kind of "inhibin" in the collecting bees, which can inhibit the synthesis of juvenile hormone in other worker bees. When inhibin is missing or insufficient, the concentration of "kinetin" (JA) in the worker bees will spontaneously rise, causing The worker bees become forager bees. Under different bee colony conditions, the development of worker bees will determine whether they develop into nurse bees normally, early or delayed due to the amount of inhibin. Later, Huang Zhiyong used experiments to verify the main predictions of this model, and the experimental results were consistent with the model (Huang and Robinson, 1996). Further experiments proved that "inhibin" is a non-volatile substance that is most effectively transmitted through direct contact between worker bees, and part of the information can also be transmitted between worker bees by feeding each other (Huang et al, 1998). Recently, the chemical component of inhibin was finally identified as ethyl oleate. Experiments have shown that ethyl oleate is many times higher in the honey sacs of collecting bees than in nurse bees, and artificially feeding ethyl oleate to worker bees can delay their collecting age (Leonciniet al, 2004). In insects, thousands of releaser pheromone molecular structures have been identified, while primer pheromone is the fourth among all animals.
The above research results were published in PNAS (1992, 89:11726-11729; 2004,101:17561-17564), Behavioral Ecology and Sociobiology (1996, 39:147-158) and Journal of comparative Physiology B (1998, 183:143-152). Among them, the 1992 PNAS article caused great repercussions in the academic community. The 1992 article was cited in J. Alcock's "Animal Behavior" textbook. At the same time, it caused many new studies in social entomology (such as computer model simulation, establishment of new models, etc.), with the number of citations reaching 108 times. The 2004 PNAS article was published as a cover story and was considered an important discovery in the field of social entomology.
2. The functional analysis of juvenile hormone in the social division of labor of bees was carried out
The behavioral development of honeybee workers is determined by the concentration of juvenile hormone (JH) in the hemolymph.
The concentration of JH in nurse bees is low, while the concentration of JH in collecting bees is high. Therefore, Huang Zhiyong conducted research on the function of JH in the social division of labor of bees and achieved the following results:
2.1. Discovered the synthesis rate of juvenile hormone Changes in JH are the main cause of changes in JH concentration
It was previously known that changes in JH concentration can affect changes in behavior, but the concentration in hemolymph may change due to synthesis, degradation or excretion. Huang Zhiyong applied the "radiochemical" method commonly used in other insects to bees for the first time, proving that the synthesis rate of bees' juvenile hormone is positively correlated with the degree of behavioral development. The research results were published in J.Insect Physiology (1991, 37:733-741). This article also laid the foundation for future determination of JH biosynthesis in bees using radiochemical methods.
2.2. Clarify the physiological mechanism of worker bee reproduction when it is not inhibited by the queen bee and larvae
Under normal circumstances, the ovaries of worker bees do not develop because they are inhibited by the queen bee and larvae. When the queen bee and larvae are absent, the worker bee's ovaries begin to develop and can also lay eggs to become drone bees. The physiological mechanism of worker bee reproduction is not clear. Huang Zhiyong's research has proven that JH is lower in the hemolymph of egg-laying worker bees than in collector bees, and at the same time, the synthesis rate of JH is also low. Ovarian development of worker bees treated with juvenile hormone was also inhibited. The article was published in General and Comparative Endocrinology (1992, 87:471-480).
2.3. Clarified the physiological mechanism of social division of labor among worker bees of the same age
In addition to the age division of labor formed by behavioral development among honey bees, there is also division of labor among worker bees of the same age, for example, Among the middle-aged worker bees, there are guard bees, corpse-cleaning bees, wax-secreting bees or storage bees. What mechanism does these bees use to enable them to have different divisions of labor even though they are the same age? The study found that guard bees and corpse-scavenger bees have high JH concentrations or synthesis rates (similar to collector bees), while wax-secreting bees and reservoir bees have low JH concentrations or synthesis rates even though they are the same age. Among the young worker bees, the JH of the nurse bees and the queen bees are the same, both are low, while among the forager bees, the JH of the pollen bees, foragers, and water bees are also the same, and they are all high. This set of experiments illustrates for the first time that JH plays a role in regulating the social division of labor among bees of the same age, especially among bees of middle age. This study is also the first time that Huang Zhiyong used highly specific antibodies to measure JH in bees using the radioimmunoassay method. This method has now become the standard method for the determination of JH concentration in bees. The research results were published in the Journal of Comparative Physiology (1994 , 174:731-739).
2.4. It was found that the juvenile hormone of worker bees changes with seasons and colony conditions
Artificially adjusting the concentration of juvenile hormone (JH) can Affects the speed of behavioral development of worker bees. However, does the JH concentration of worker bees in normal bees change with other conditions, such as season and colony conditions? Experiments show that after collector bees stop collecting in autumn, as the temperature decreases, the concentration of worker bees changes. The JH concentration and synthesis rate of both bees and young bees will decrease. When the bee colonies are artificially moved to the freezer in summer, JH also decreases significantly on the 8th day; this indicates that the bees may adjust based on temperature rather than light cycle. JH. The JH of worker bees rises again in the spring and eventually becomes the collector bee. The study found that changes in the colony conditions can also adjust the concentration of JH in the same colony (all worker bees emerge on the same day, singlecohortcolony), in which the worker bees do not become precocious. Before collecting bees, the concentration of JH increases significantly. In addition, when the bee colony is crowded (a prerequisite for bee colony division), the increase in JH concentration is delayed, which delays the development of worker bees that are about to divide. The worker bees in the colony are physiologically younger, so they can adapt to the situation that the worker bees in the new colony need to nurse the young bees for a longer time.
The above research results were published in the Journal of Comparative Physiology B (1995, 165:18-28), Journal of Insect Physiology (2000, 46:243-249) and Journal ofEconomic Entomology (2005, 98:274-278).
2.5. Clarified the relationship between the concentration of juvenile hormone and the vibrancy and ferocity of the dance
As mentioned above, juvenile hormone (JH) can be produced under the influence of many factors change. Worker bees have a vibrational dance, which mainly serves as a call to action, allowing the vibrated worker bees to increase their workload. This behavior occurs mainly when the weather improves after a long period of rain, or before flying in groups. Increase. Huang Zhiyong imagined that this dance might be able to quickly increase the concentration of JH, thereby increasing the activity level of worker bees. Facts have proved that Huang Zhiyong's hypothesis is correct. Within 15-30 minutes after the worker bees were vibrated, the JH concentration was significantly higher than that of the control bees (which were not vibrated within 5-30 minutes). Huang Zhiyong also speculated that the increase in JH may be related to the ferocity of the worker bees. The study found that both winter bees and summer nurse bees have low JH concentrations, and the rate of resistance behavior to foreign bees is also low. In the same bee colony in summer, the JH concentration of bees that are aggressive toward foreign bees is also higher than that of non-ferocious bees. Therefore, JH is positively correlated with the ferocity of bees. The research results were published in J. Insect Physiology (2001, 47:1243-1247) and Ethology (2004,110:977-985).
The above research results clarify the causes and consequences of increased JH concentration, and play a significant role in understanding the social behavior of bees.
3. Conducted research on the impact of genetically modified plant pollen on bees and their pests
Genetically modified plants are increasingly planted around the world, but most of them are harmful to bees, an important Assessment of beneficial insect effects is limited to laboratory assays using proteins. Huang Zhiyong believes that since the inserted gene may have multiple effects, or alternative splicing, or the interaction between the host and the inserted gene, it may be more reliable to directly use pollen to measure the impact of transgenic plants on bees. Huang Zhiyong measured the effects of genetically modified corn (Cry1A(b), Cry1F) pollen and genetically modified (herbicide-resistant) rapeseed (canola) on bees. Experiments showed that in field experiments, genetically modified corn pollen affected the survival rate of bee larvae and pupae. , pupal weight, and hemolymph protein concentration of newly emerged bees had no significant side effects. On the contrary, Cry1F pollen has a very good control effect on Galleria mellonella, a bee pest (killing rate is 100%). In two years of field experiments, Huang Zhiyong found no side effects of transgenic rapeseed pollen on the larval survival rate, pupal survival rate, pupa weight of bees, or the hemolymph protein concentration of newly emerged bees. These experiments propose a new assessment model that will influence future more comprehensive assessments of transgenic plants. The research results were published in J.Apicultural Research (2003, 42:77-78) and J. Economic Entomology (2004, 97:1517-1523).
4. The resistance mechanism of Horny Varroa to pyrethroids, its molecular classification in China, and research on its prevention and control
The Horny Varroa is a recognized and worldwide pest of honeybees. The biggest pest in the world. In recent years, giant varroa mites have developed resistance to a pyrethrin (fluvalinate, fluvalinate) that has been used since 1991, but the resistance mechanism is unclear. Because the target of all pyrethroid lipid drugs is sodium ion channels, Huang Zhiyong hypothesized that the resistance of Varroa mites is due to genetic changes in sodium ion channels, thereby changing the binding of sodium ion channel proteins to pyrethroid lipids. After a long period of hard work, the research teams of Dong Ke and Huang Zhiyong collaborated to clone and sequence the entire gene of the sodium ion channel in an arachnid for the first time. The Varroa mite sodium channel (VmNa) cDNA has 6645 bases and 2215 amino acids. The calculated amino acid sequence is 51% similar to Drosophila and 41% similar to the Nav1.6 gene of mammals. They found that four mutations may be related to the drug resistance of Varroa mites, namely F758L, L826P, I982V and M1055I (the numbers are the amino acid positions, the first letter is the original amino acid, and the last letter is the mutated amino acid).
After years of hard work, the sodium channel gene of the Varroa mite was finally expressed in frog eggs (zenopus), thus laying the foundation for further in vitro determination of its pharmacological properties. The results were published in J.Apicultural Research (2002, 40:17-25) and Insect Biochemistry and Molecular Biology (2003, 33: 733-739).
Working with domestic (Zhou Ting, Huang Shuangxiu) and Australian scientists (Denis Anderson), we conducted a taxonomic study on the giant varroa mite using mitochondrial DNA molecular markers, and for the first time identified the differences between Western honey bee and Apis mellifera in China. The Varroa destructor found on Oriental bees is Varroa destructor, but Varroa jacobsoni, which is generally thought to harm Oriental bees, does not exist at all. They also discovered a new genotype, named China2. These results laid a good foundation for the prevention, control and quarantine of giant varroa mites in China in the future. The results were published in Apidologie (2004, 35: 645-654).
It has been known for a long time that the giant Varroa mite prefers drone larvae, and its preference for drone larvae and worker bee larvae is about 10:1. Many beekeepers put the drone's spleen into the hive, take it out after sealing it, and put it in the freezer to kill the drone and the large mites on it. Huang Zhiyong invented a device to kill giant hornet mites in 2002 and has obtained a U.S. patent (Patent No. #6,475,061). Recently, companies have purchased the manufacturing rights to patents. The results were published in AmericanBee Journal (2001, 141: 730-732).
5. Evolutionary ecological study of bee sex alleles
The sex of bees is determined by a "complementary sex determinative" (csd) mechanism. The diploid heterozygote becomes female, and the haploid becomes male. However, when the sex allele is homozygous, the diploid still develops into a male. In a normal bee colony, diploid males are eaten by worker bees. Therefore, the selection pressure to make worker bees heterozygous is strong. For more than a hundred years, this theory has been nothing more than a hypothesis. Only recently was the gene for csd sequenced and finally proven to be a sex-determining gene (Beye et al, 2003). Due to strong selection pressure, csd should show a high degree of intraspecific polymorphism (intraspecific polymorphism), and the age of its alleles may be older than the age of the species. Huang and his collaborators compared the csd of three species of bees (Apis mellifera, Apis orientalis, and Apis mellifera) with the gene sequences of randomly selected neutral gene regions, and found that: ⑴. The polymorphisms of csd are higher than the polymorphisms of neutral genes. The sex is about seven times higher; ⑵. Gene family tree analysis found that csd has intraspecific polymorphism, but the neutral gene does not; ⑶. In line with the prediction of low-frequency allele advantage (rareallele advantage), non-synonymous mutations (non -synonymous mutation) was under positive selection pressure when it first appeared. ⑷. It was found that among three different species of bees, each species has its own hyper-variable repeat region, and it is speculated that each species has its own different mechanism that determines allele specificity. The research paper was published together in a special issue of Bee Genome in 2006 (Genome Research 16: 1366-1375)