Standard methods for small hive beetle research.

Authors: Peter Neumann, Jay D Evans, Jeff S Pettis, Christian W W Pirk, Marc O Schäfer, Gina Tanner, James D Ellis.

Table of contents

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Thanks to: Luis Gerardo Lara Alvarez- Translation. Jose Antonio Dorantes Ugalde, Luz Maria Saldaña Loza- Reviewing

1. Introduction
2. Diagnostics and sampling
   2.1. Beetle appearance during the different developmental stages

      2.1.1. Eggs
      2.1.2. Larvae
      2.1.3. Pupae
      2.1.4. Adults
   2.2. Beetle locations during the different development stages

      2.2.1. Eggs
      2.2.2. Larvae
      2.2.3. Pupae
      2.2.4. Adults
   2.3. Shipped bee samples
      2.3.1. Inspection of honey bee queen shipments for small hive beetle
   2.4. Sampling of adult small hive beetles
      2.4.1. Manual collection
      2.4.2. Traps in colonies
      2.4.3. Traps outside of colonies
 Trapping of adult small hive beetles Trapping of small hive beetle larvae
      2.4.4. Storage and shipment of SHB samples
      2.4.5. Molecular diagnostics
 DNA extraction Mitochondrial DNA analyses Microsatellite analyses Screening hive debris for SHB Future genetic work with SHB
3. Techniques for experimental investigations
   3.1. Laboratory techniques
      3.1.1. Maintaining adult SHBs in the laboratory and transport
 SHB diet SHB maintenance and transport conditions Mass maintenance Individual maintenance
      3.1.2. Rearing techniques Rearing Procedure Individual rearing approach
      3.1.3. Sex determination Sex determination of adults Sex determination of pupae
      3.1.4. Marking techniques Marking SHB larvae Marking adult SHB
   Feeding adult SHBs with coloured food
   Thoracic notching of adult SHBs
      3.1.5. Effect of external conditions on SHB in the laboratory Investigating diet effects on SHB reproduction Determining soil effects on SHB pupation
      3.1.6. Transmission of bee pathogens by small hive beetles Investigating potential interactions between A. tumida and Paenibacillus larvae, the causative
                     organism of American foulbrood
 Investigating the potential of SHB to vector honey bee viruses
      3.1.7. Investigations of SHB pathogens and parasites Inoculating SHB larvae with entomopathogenic fungi Infesting SHBs with nematodes
   In vitro infestation of wandering larvae with an aqueous suspension of nematodes
   In vitro soil infestation of larvae using a sand bioassay
   In vitro soil infestation of pupae with an aqueous suspension of nematodes Autoclave soil.
   In vitro soil infestation of pupae with nematode-infected cadavers
   3.2. Field techniques
      3.2.1. Investigating intra-colonial interactions between adult bees and adult SHB
 General experimental establishment Behavioural categories Investigating behavioural interactions of adult honey bees and adult SHBs at the colony entrance – two
                     alternative options

      3.2.2. Investigating SHB oviposition behaviour and bee hygienic responses to SHB eggs and young larvae Promoting SHB oviposition behaviour in capped bee brood Identifying and marking brood cells in which SHB have oviposited Determining honey bee hygienic responses to SHB eggs and young larvae. Determining proportion of perforated capped brood cells containing SHB eggs (oviposition rate)
      3.2.3. Determining the number and distribution of adult SHBs inside a field colony and winter clusters
               and starting colonies without SHBs
 Visually screening the number of beetles in live colonies and/or removing beetles to
                     start beetle-free colonies
 Counting the number and distribution of beetles in freshly-killed colonies
   Killing with liquid nitrogen
   Killing with petrol fuel
   Freeze killing Counting the number of beetles and defining their spatial distribution in winter clusters killed by
                     dipping in liquid nitrogen

      3.2.4. Collecting SHB eggs
      3.2.5. Introducing adult SHB into colonies
4. Acknowledgments
5. References