Tropilaelaps species identification and viral load evaluation of Tropilaelaps and Varroa mites and their Apis mellifera hosts in Palawan, Philippines.

Apis mellifera pupae and their parasites Tropilaelaps and Varroa destructor were collected from honey bee hives in Palawan, Philippines for species identification of the Tropilaelaps and viral analyses. Genetic analysis identified Tropilaelaps mercedesae infesting A. mellifera on the island. Viral analyses showed that all pupae and their infesting Tropilaelaps or Varroa shared the same Deformed Wing Virus (DWV) variant infections with DWV-B being more prevalent than DWV-A. Pupae infested with either Varroa or Tropilaelaps had higher levels of both DWV variants than uninfested pupae. Vigilance is needed to prevent the spread of Tropilaelaps clareae into Palawan and T. mercedesae and DWV variants from Palawan to other provinces.

Feeding by Tropilaelaps mercedesae on pre- and post-capped brood increases damage to Apis mellifera colonies.

Tropilaelaps mercedesae parasitism can cause Apis mellifera colony mortality in Asia. Here, we report for the first time that tropilaelaps mites feed on both pre- and post-capped stages of honey bees. Feeding on pre-capped brood may extend their survival outside capped brood cells, especially in areas where brood production is year-round. In this study, we examined the types of injury inflicted by tropilaelaps mites on different stages of honey bees, the survival of adult honey bees, and level of honey bee viruses in 4th instar larvae and prepupae. The injuries inflicted on different developing honey bee stages were visualised by staining with trypan blue. Among pre-capped stages, 4th instar larvae sustained the highest number of wounds (4.6 ± 0.5/larva) while 2nd-3rd larval instars had at least two wounds. Consequently, wounds were evident on uninfested capped brood (5th-6th instar larvae = 3.91 ± 0.64 wounds; prepupae = 5.25 ± 0.73 wounds). Tropilaelaps mite infestations resulted in 3.4- and 6-fold increases in the number of wounds in 5th-6th instar larvae and prepupae as compared to uninfested capped brood, respectively. When wound-inflicted prepupae metamorphosed to white-eyed pupae, all wound scars disappeared with the exuviae. This healing of wounds contributed to the reduction of the number of wounds (≤10) observed on the different pupal stages. Transmission of mite-borne virus such as Deformed Wing Virus (DWV) was also enhanced by mites feeding on early larval stages. DWV and Black Queen Cell Virus (BQCV) were detected in all 4th instar larvae and prepupae analysed. However, viral levels were more pronounced in scarred 4th instar larvae and infested prepupae. The remarkably high numbers of wounds and viral load on scarred or infested developing honey bees may have caused significant weight loss and extensive injuries observed on the abdomen, wings, legs, proboscis and antennae of adult honey bees. Together, the survival of infested honey bees was significantly compromised. This study demonstrates the ability of tropilaelaps mites to inflict profound damage on A. mellifera hosts. Effective management approaches need to be developed to mitigate tropilaelaps mite problems.

Comb Irradiation Has Limited, Interactive Effects on Colony Performance or Pathogens in Bees, Varroa destructor and Wax Based on Two Honey Bee Stocks.

Parasitic mites and pathogens compromise honey bee health. Development of sustainable and integrative methods of managing these problems will minimize their detrimental impact on honey bees. Here, we aimed to determine if the combination of using mite-resistant stocks along with gamma-irradiated combs influences colony health and productivity. The major finding concerned honey bee genotype confirming that Russian honey bees are more resistant to Varroa destructor than Italian honey bees. The effect of comb irradiation was inconsistent showing a significant increase in adult bee population and amount of stored pollen in 2015, but not in 2016. The increased amount of stored pollen was probably associated with larger adult population in colonies with irradiated combs in September 2015 regardless of honey bee stock. Nevertheless, the ability of bees to collect and store more pollen in the irradiated group does not appear to compensate the negative impacts of mite parasitism on honey bees especially in the Italian bees, which consistently suffered significant colony losses during both years. Results of viral analyses of wax, newly emerged bees, and Varroa and their pupal hosts showed common detections of Deformed wing virus (DWV), Varroa destructor virus (VDV-1), Chronic bee paralysis virus (CBPV), and Black queen cell virus (BQCV). Wax samples had on average ~4 viruses or pathogens detected in both irradiated and non-irradiated combs. Although pathogen levels varied by month, some interesting effects of honey bee stock and irradiation treatment were notable, indicating how traits of mite resistance and alternative treatments may have additive effects. Further, this study indicates that wax may be a transmission route of viral infection. In addition, pupae and their infesting mites from Italian colonies exhibited higher levels of DWV than those from Russian colonies suggesting potential DWV resistance by Russian honey bees. CBPV levels were also reduced in mites from Russian colonies in general and in mites, mite-infested pupae, and newly emerged bees that were collected from irradiated combs. However, BQCV levels were not reduced by comb irradiation. Overall, the contribution of irradiating comb in improving honey bee health and colony survival appears to be subtle, but may be useful as part of an integrated pest management strategy with the addition of using mite-resistant stocks.

Successful reproduction of unmated Tropilaelaps mercedesae and its implication on mite population growth in Apis mellifera colonies.

Successful reproduction by unmated Tropilaelaps mercedesae is reported here for the first time. Of the eight mature daughters that did not have male mates within their natal cells, four produced both mature sons and daughters, and four produced mature daughters only. Overall, 78% of the new daughters that had no egg-laying experience, and 84% of the foundresses that had or had not laid previously reproduced. Both inoculum daughter and foundress mites were collected from tan-bodied pupae and inoculated immediately. Therefore, our results suggest that phoresy is not required for reproduction in tropilaelaps mites. The ability of virgin females to lay both males and females (deuterotoky), and to reproduce without spending a phoretic period on adult bees may play major roles in tropilaelaps mites' competitive advantage over varroa mites in Apis mellifera colonies.

Comparative Flight Activities and Pathogen Load of Two Stocks of Honey Bees Reared in Gamma-Irradiated Combs.

Gamma irradiation is known to inactivate various pathogens that negatively affect honey bee health. Bee pathogens, such as Deformed wing virus (DWV) and Nosema spp., have a deleterious impact on foraging activities and bee survival, and have been detected in combs. In this study, we assessed the effects of gamma irradiation on the flight activities, pathogen load, and survival of two honey bee stocks that were reared in irradiated and non-irradiated combs. Overall, bee genotype influenced the average number of daily flights, the total number of foraging flights, and total flight duration, in which the Russian honey bees outperformed the Italian honey bees. Exposing combs to gamma irradiation only affected the age at first flight, with worker bees that were reared in non-irradiated combs foraging prematurely compared to those reared in irradiated combs. Precocious foraging may be associated with the higher levels of DWV in bees reared in non-irradiated combs and also with the lower amount of pollen stores in colonies that used non-irradiated combs. These data suggest that gamma irradiation of combs can help minimize the negative impact of DWV in honey bees. Since colonies with irradiated combs stored more pollen than those with non-irradiated combs, crop pollination efficiency may be further improved when mite-resistant stocks are used, since they performed more flights and had longer flight durations.

An evaluation of the associations of parameters related to the fall of Varroa destructor (Acari: Varroidae) from commercial honey bee (Hymenoptera: Apidae) colonies as tools for selective breeding for mite resistance.

Varroa destructor (Anderson and Trueman) trapped on bottom boards were assessed as indirect measurements of colony mite population differences and potential indicators of mite resistance in commercial colonies of Russian and Italian honey bees (Apis mellifera L.) by using 35 candidate measurements. Measurements included numbers of damaged and nondamaged younger mites, nymphs, damaged and nondamaged older mites, fresh mites, and all mites, each as a proportion of total mites in the colonies and as a proportion of all trapped mites or all trapped fresh mites. Several measurements differed strongly between the stocks, suggesting that the detailed characteristics of trapped mites may reflect the operation of resistance mechanisms in the Russian honey bees. Regression analyses were used to determine the relationships of these candidate measurements with the number of mites in the colonies. The largest positive regressions differed for the two stocks (Italian honey bees: trapped mites and trapped younger mites; Russian honey bees: trapped younger mites and trapped fresh mites). Also, the regressions for Italian honey bees were substantially stronger. The largest negative regressions with colony mites for both stocks were for the proportion of older mites out of all trapped mites. Although these regressions were statistically significant and consistent with those previously reported, they were weaker than those previously reported. The numbers of mites in the colonies were low, especially in the Russian honey bee colonies, which may have negatively influenced the precision of the regressions.

Functionality of Varroa-resistant honey bees (Hymenoptera: Apidae) when used for western U.S. honey production and almond pollination.

Two types of honey bees, Apis mellifera L., bred for resistance to Varroa destructor Anderson & Trueman, were evaluated for performance when used for honey production in Montana, and for almond pollination the following winter. Colonies of Russian honey bees and outcrossed honey bees with Varroa-sensitive hygiene (VSH) were compared with control colonies of Italian honey bees. All colonies were managed without miticide treatments. In total, 185 and 175 colonies were established for trials in 2010-2011 and 2011-2012, respectively. Survival of colonies with original queens or with supersedure queens was similar among stocks for both years. Colony sizes of the Varroa-resistant stocks were as large as or larger than the control colonies during periods critical to honey production and almond pollination. Honey production varied among stocks. In the first year, all stocks produced similar amounts of honey. In the second year, Russian honey bees colonies produced less honey than the control colonies. V. destructor infestations also varied among stocks. In the first year, control colonies had more infesting mites than either of the Varroa-resistant stocks, especially later in the year. In the second year, the control and outcrossed Varroa-sensitive hygiene colonies had high and damaging levels of infestation while the Russian honey bees colonies maintained lower levels of infestation. Infestations of Acarapis woodi (Rennie) were generally infrequent and low. All the stocks had similarly high Nosema ceranae infections in the spring and following winter of both years. Overall, the two Varroa-resistant stocks functioned adequately in this model beekeeping system.

Phenotypic and genetic analyses of the varroa sensitive hygienic trait in Russian honey bee (hymenoptera: apidae) colonies.

Varroa destructor continues to threaten colonies of European honey bees. General hygiene, and more specific Varroa Sensitive Hygiene (VSH), provide resistance towards the Varroa mite in a number of stocks. In this study, 32 Russian (RHB) and 14 Italian honey bee colonies were assessed for the VSH trait using two different assays. Firstly, colonies were assessed using the standard VSH behavioural assay of the change in infestation of a highly infested donor comb after a one-week exposure. Secondly, the same colonies were assessed using an "actual brood removal assay" that measured the removal of brood in a section created within the donor combs as a potential alternative measure of hygiene towards Varroa-infested brood. All colonies were then analysed for the recently discovered VSH quantitative trait locus (QTL) to determine whether the genetic mechanisms were similar across different stocks. Based on the two assays, RHB colonies were consistently more hygienic toward Varroa-infested brood than Italian honey bee colonies. The actual number of brood cells removed in the defined section was negatively correlated with the Varroa infestations of the colonies (r2 = 0.25). Only two (percentages of brood removed and reproductive foundress Varroa) out of nine phenotypic parameters showed significant associations with genotype distributions. However, the allele associated with each parameter was the opposite of that determined by VSH mapping. In this study, RHB colonies showed high levels of hygienic behaviour towards Varroa -infested brood. The genetic mechanisms are similar to those of the VSH stock, though the opposite allele associates in RHB, indicating a stable recombination event before the selection of the VSH stock. The measurement of brood removal is a simple, reliable alternative method of measuring hygienic behaviour towards Varroa mites, at least in RHB stock.

Observations on the removal of brood inoculated with Tropilaelaps mercedesae (Acari: Laelapidae) and the mite's reproductive success in Apis mellifera colonies.

This study assessed the response of Apis mellifera to brood deliberately infested with Tropilaelaps mercedesae. The reproductive success of T. mercedesae in mite-inoculated and naturally infested brood was also compared. The presence of T. mercedesae inside brood cells significantly affected brood removal. Thai A. mellifera removed 52.6 ± 8.2 % of the brood inoculated with T. mercedesae as compared to 17.2 ± 1.8 and 5.7 ± 1.1 % removal rates for the groups of brood with their cell cappings opened and closed without mite inoculation and the control brood (undisturbed, no mite inoculation), respectively. Brood removal peaked during the second and third days post inoculation when test brood was at the prepupal stage. Overall, non-reproduction (NR) of foundress T. mercedesae was high. However, when NR was measured based on the criteria used for Varroa, the naturally infested pupae (NIP) supported the highest NR (92.8 %). Newly sealed larvae inoculated with Tropilaelaps collected from newly sealed larvae (NSL) had 78.2 % NR and those inoculated with Tropilaelaps collected from tan-bodied pupae (TBP) had 76.8 % NR. Since Tropilaelaps is known to have a short development period and nearly all progeny reach adulthood by the time of host emergence, we also used two Tropilaelaps-specific criteria to determine NR. Foundresses that did not produce progeny and those that produced only one progeny were considered NR. Using these two criteria, NR decreased tremendously but showed similar trends with means of 65, 40 and 33 % for NIP, NSL and TBP, respectively. High NR in the NIP group may indicate increased hygienic behavior in Thai A. mellifera colonies. The removal of infested prepupae or tan-bodied pupae will likely decrease the reproductive potential of Tropilaelaps. Our study suggests that brood removal may be one of the resistance mechanisms towards T. mercedesae by naturally adapted Thai A. mellifera.

Associations of parameters related to the fall of Varroa destructor (Mesostigmata: Varroidae) in Russian and Italian honey bee (Hymenoptera: Apidae) colonies.

Varroa destructor (Anderson and Truman) trapped on bottom boards were assessed as indirect measurements of colony mite populations and mite fall in colonies of Russian and Italian honey bees using 29 candidate measurements. Measurements included damaged and nondamaged younger mites, damaged and nondamaged older mites, fresh mites and all mites, each as a proportion of total mites in the colonies and as a proportion of all trapped mites or all trapped fresh mites. Regression analyses were used to determine the relationships of these candidate measurements to the number of mites in the colonies. The largest positive regressions were found for trapped younger mites (Y) and trapped fresh mites (F). Measurments of Y and F across time could be used to estimate mite population growth for the purposes of selective breeding. The largest negative regressions with colony mites were observed for: trapped older mites/trapped mites (O/T), trapped older mites/trapped younger mites (O/Y), and trapped injured older mites/injured mites (IO/I). O/T and O/Y are significantly higher for Russian honey bee colonies suggesting that they are related to at least some of the mechanisms used by Russian honey bee to resist Varroa population growth. O/T and O/Y have strong negative relationships with colony mites for both Russian honey bee and Italian colonies suggesting that both strains possibly could be selected for reduced colony mites using O/T or O/Y.

Age and reproductive status of adult Varroa mites affect grooming success of honey bees.

This study evaluated for the first time the grooming response of honey bees to Varroa mites of different ages and reproductive statuses in the laboratory. Plastic cages containing a section of dark comb and about 200 bees were inoculated with groups of four classes of mites: gravid, phoretic foundresses, phoretic daughters and a combination of gravid and phoretic foundress mites. Each cage received 20 mites belonging to one of these classes. Our results showed that, 1 day after mite inoculation, phoretic daughter mites were the most prone to grooming by honey bees with an average mite drop of 49.8 ± 2.6 %. The lowest mite drop was recorded for bees inoculated with phoretic foundresses (30.3 ± 3.6 %) but was comparable to bees inoculated with gravid mites (31.8 ± 3.8 %) and the combination of gravid and phoretic foundress mites (34.2 ± 3.2 %). No differences among mite types were detected during the second and third days of observation. Regardless of mite type, the highest mite drop was recorded on the first day (35 ± 2.1 %) compared to the drop for any subsequent day (<10 %). Because of the great reproductive potential of daughter mites, their inclusion in assessments of grooming behaviour may increase our insight into the importance of grooming in mite resistance.

Asynchronous development of honey bee host and Varroa destructor (Mesostigmata: Varroidae) influences reproductive potential of mites.

A high proportion of nonreproductive (NR) Varroa destructor Anderson & Trueman (Mesostigmata: Varroidae), is commonly observed in honey bee colonies displaying the varroa sensitive hygienic trait (VSH). This study was conducted to determine the influence of brood removal and subsequent host reinvasion of varroa mites on mite reproduction. We collected foundress mites from stages of brood (newly sealed larvae, prepupae, white-eyed pupae, and pink-eyed pupae) and phoretic mites from adult bees. We then inoculated these mites into cells containing newly sealed larvae. Successful reproduction (foundress laid both a mature male and female) was low (13%) but most common in mites coming from sealed larvae. Unsuccessful reproductive attempts (foundress failed to produce both a mature male and female) were most common in mites from sealed larvae (22%) and prepupae (61%). Lack of any progeny was most common for mites from white-eyed (83%) and pink-eyed pupae (92%). We also collected foundress mites from sealed larvae and transferred them to cells containing newly sealed larvae, prepupae, white-eyed pupae, or pink-eyed pupae. Successful reproduction only occurred in the transfers to sealed larvae (26%). Unsuccessful reproductive attempts were most common in transfers to newly sealed larvae (40%) and to prepupae (25%). Unsuccessful attempts involved the production of immature progeny (60%), the production of only mature daughters (26%) or the production of only a mature male (14%). Generally, lack of progeny was not associated with mites having a lack of stored sperm. Our results suggest that mites exposed to the removal of prepupae or older brood due to hygiene are unlikely to produce viable mites if they invade new hosts soon after brood removal. Asynchrony between the reproductive status of reinvading mites and the developmental stage of their reinvasion hosts may be a primary cause of NR mites in hygienic colonies. Even if reinvading mites use hosts having the proper age for infestation, only a minority of them will reproduce.

Effect of height and color on the efficiency of pole traps for Aethina tumida (Coleoptera: Nitidulidae).

Olfactory cues released by adult bees, brood, pollen, and honey from a honey bee, Apis mellifera L., colony are the primary stimuli that guide the beetle Aethina tumida Murray (Coleoptera: Nitidulidae) to host colonies. To investigate the response of adult A. tumida to visual stimuli, we tested the influence of color and height on trap efficiency. Two pole trap colors (black and white) were evaluated at three heights (46 cm, 1 m, and 3m) from October 2008 to December 2009. A. tumida were trapped in the greatest numbers between 17 April and 15 May 2009. The lowest numbers were captured during the winter and fall. The trapping results showed that both color and trap height significantly influenced capture. The average catch in the white traps (mean +/- SE, 2.47 +/- 0.30) was significantly higher than that of the black traps (1.53 +/- 0.29) probably because white is more reflective than black. Among the heights evaluated, there were more beetles caught when traps were positioned at 46 cm (the same height as the entrance of the hives) with 3.07 +/- 0.51 beetles compared with beetles captured at 1 m (1.88 +/- 0.30) or 3 m (1.06 +/- 0.18) high. Male and female beetles exhibited similar responses to trap color and height. The relationship between the numbers of beetles in colonies and capture rates in traps was very poor and did not provide a basis to evaluate trap efficiency. In addition, because capture rates seemed generally low in relationship to the number of beetles in the apiary, substantial improvements to the trap may be necessary.

Comparative resistance of Russian and Italian honey bees (Hymenoptera: Apidae) to small hive beetles (Coleoptera: Nitidulidae).

To compare resistance to small hive beetles (Coleoptera: Nitidulidae) between Russian and commercial Italian honey bees (Hymenoptera: Apidae), the numbers of invading beetles, their population levels through time and small hive beetle reproduction inside the colonies were monitored. We found that the genotype of queens introduced into nucleus colonies had no immediate effect on small hive beetle invasion. However, the influence of honey bee stock on small hive beetle invasion was pronounced once test bees populated the hives. In colonies deliberately freed from small hive beetle during each observation period, the average number of invading beetles was higher in the Italian colonies (29 +/- 5 beetles) than in the Russian honey bee colonies (16 +/- 3 beetles). A similar trend was observed in colonies that were allowed to be freely colonized by beetles throughout the experimental period (Italian, 11.46 +/- 1.35; Russian, 5.21 +/- 0.66 beetles). A linear regression analysis showed no relationships between the number of beetles in the colonies and adult bee population (r2 = 0.1034, P = 0.297), brood produced (r2 = 0.1488, P = 0.132), or amount of pollen (P = 0.1036, P = 0.295). There were more Italian colonies that supported small hive beetle reproduction than Russian colonies. Regardless of stock, the use of entrance reducers had a significant effect on the average number of small hive beetle (with reducer, 16 +/- 3; without reducer, 27 +/- 5 beetles). However, there was no effect on bee population (with reducer, 13.20 +/- 0.71; without reducer, 14.60 +/- 0.70 frames) or brood production (with reducer, 6.12 +/- 0.30; without reducer, 6.44 +/- 0.34 frames). Overall, Russian honey bees were more resistant to small hive beetle than Italian honey bees as indicated by fewer invading beetles, lower small hive beetle population through time, and lesser reproduction.

Comparative reproduction of Varroa destructor in different types of Russian and Italian honey bee combs.

Earlier studies showed that Russian honey bees support slow growth of varroa mite population. We studied whether or not comb type influenced varroa reproduction in both Russian and Italian honey bees, and whether Russian bees produced comb which inhibited varroa reproduction. The major differences found in this study concerned honey bee type. Overall, the Russian honey bees had lower (2.44 +/- 0.18%) levels of varroa infestation than Italian honey bees (7.20 +/- 0.60%). This decreased infestation resulted in part from a reduced number of viable female offspring per foundress in the Russian (0.85 +/- 0.04 female) compared to the Italian (1.23 +/- 0.04 females) honey bee colonies. In addition, there was an effect by the comb built by the Russian honey bee colonies that reduced varroa reproduction. When comparing combs having Russian or Italian colony origins, Russian honey bee colonies had more non-reproducing foundress mites and fewer viable female offspring in Russian honey bee comb. This difference did not occur in Italian colonies. The age of comb in this study had mixed effects. Older comb produced similar responses for six of the seven varroa infestation parameters measured. In colonies of Italian honey bees, the older comb (2001 dark) had fewer (1.13 +/- 0.07 females) viable female offspring per foundress than were found in the 2002 new (1.21 +/- 0.06 females) and 1980s new (1.36 +/- 0.08 females) combs. This difference did not occur with Russian honey bee colonies where the number of viable female offspring was low in all three types of combs. This study suggests that honey bee type largely influences growth of varroa mite population in a colony.